bluuebunny/crossref_metadata_2025 huggingface dataset:
Environmental Impact Evaluation, Land Use Planning, and the Housing Consumer*
Authors: James Franklin J. , Muller Thomas
Published: October 2003

A number of states are following the federal government's lead and requiring environmental impact reviews of new development proposals. This paper examines ambitious EIR programs in Florida and California with the aim of assessing the costs and benefits of their programs, and, by extension, of state‐mandated EIR programs in general. The focus is on new housing production. The findings are that EIR shows some promise of enabling communities to better protect themselves from adverse side effects of new development. At the same time, EIR adds what appears to be a modest increment to the overall costs of new housing. However, because EIR has expanded local government planning options, one of the side effects of EIR is likely to be the enhanced ability of localities to exclude moderate income families from new housing.
Transnationalism and identity: a tale of two faces and multiple lives
Authors: Ghosh Sutama , Wang Lu
Published: October 2003

Transnational ties add new complexities to the continuous and dynamic processes of identity formation. Based on self‐reflexive narratives, this paper examines the authors’ identity transformations associated with their respective transnational experiences. We are two Asian women from India and China, respectively, who are international students pursuing doctoral degrees in Canada. Although we share similar demographic and economic backgrounds, we perform distinct transnational acts. Focusing specifically on social and cultural linkages, we have identified reasons that have influenced our cross‐border involvements. Based on our findings, we present an emergent conceptual framework that highlights interactive psychological, sociocultural and economic processes that influence the formation of individual transnational identities. We also share with our readers some methodological lessons learnt along our path of self‐expression, analysis and representation.
The Central Nucleus of the Amygdala Modulates Gut‐Related Neurons in the Dorsal Vagal Complex in Rats
Authors: Zhang Xueguo , Cui Jinjuan , Tan Zhenjun , Jiang Chunhui , Fogel Ronald
Published: October 2003

Using retrograde tract‐tracing and electrophysiological methods, we characterized the anatomical and functional relationship between the central nucleus of the amygdala and the dorsal vagal complex. Retrograde tract‐tracing techniques revealed that the central nucleus of the amygdala projects to the dorsal vagal complex with a topographic distribution. Following injection of retrograde tracer into the vagal complex, retrogradely labelled neurons in the central nucleus of the amygdala were clustered in the central portion at the rostral level and in the medial part at the middle level of the nucleus. Few labelled neurons were seen at the caudal level. Electrical stimulation of the central nucleus of the amygdala altered the basal firing rates of 65 % of gut‐related neurons in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus. Eighty‐one percent of the neurons in the nucleus of the solitary tract and 47 % of the neurons in the dorsal motor nucleus were inhibited. Electrical stimulation of the central nucleus of the amygdala also modulated the response of neurons in the dorsal vagal complex to gastrointestinal stimuli. The predominant effect on the neurons of the nucleus of the solitary tract was inhibition. These results suggest that the central nucleus of the amygdala influences gut‐related neurons in the dorsal vagal complex and provides a neuronal circuitry that explains the regulation of gastrointestinal activity by the amygdala.
The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms
Authors: Linari Marco , Bottinelli Roberto , Pellegrino Maria Antonietta , Reconditi Massimo , Reggiani Carlo , Lombardi Vincenzo
Published: October 2003

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca2+ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single‐fibre gel electrophoresis. Fibres were activated by using a new set‐up that allows development of most of the tension following a temperature jump from 0–1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere non‐uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40–70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force‐generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin–myosin interactions under stretch become independent of the MHC isoform.
Small‐ and Intermediate‐Conductance Calcium‐Activated K+ Channels Provide Different Facets of Endothelium‐Dependent Hyperpolarization in Rat Mesenteric Artery
Authors: Crane G. J. , Gallagher N. , Dora K. A. , Garland C. J.
Published: October 2003

Activation of both small‐conductance (SKCa) and intermediate‐conductance (IKCa) Ca2+‐activated K+ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K+ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration‐dependent smooth muscle hyperpolarization, increasing the resting potential (approx. −53 mV) by around 20 mV at 3 μm. Similar hyperpolarization was evoked with cyclopiazonic acid (10 μm, an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1‐EBIO (300 μm, an IKCa activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 nm, an SKCa blocker) but was unaltered by 1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole (1 μm TRAM‐34, an IKCa blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around −70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential (circa−58 mV). TRAM‐34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh‐evoked hyperpolarization was completely abolished. These data suggest that true endothelium‐dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SKCa channels, whereas IKCa channels play an important role during the ACh‐mediated repolarization phase only observed following depolarization.
Long Vasodilator Reflexes Projecting Through the Myenteric Plexus in Guinea‐Pig Ileum
Authors: Reed David E. , Vanner Stephen J.
Published: October 2003

This study examined enteric neural reflexes activating submucosal cholinergic vasodilator motoneurons, which innervate the final resistance vessels regulating mucosal blood flow. Videomicroscopy was employed to monitor dilatation of submucosal arterioles in in vitro preparations from guinea‐pig ileum. Balloon distension of intact lumen evoked reflex vasodilatation and flat sheet preparations were employed to separate mucosal mechanical stimulation from intestinal distension. Mucosal stroking and balloon distension of the orad segment evoked vasodilatations > 1.5 cm from the stimulating site. Mucosal stimulation was blocked by combined 5‐HT3/5HT4 antagonists but distension‐evoked responses were unaffected. Distension‐evoked responses were also unaffected by nifedipine (5 μm) or nifedipine (1 μm) and wortmannin (300 nm), suggesting stretch activation rather than stretch‐activated contraction was involved. Mucosal and distension‐evoked responses were completely blocked when the myenteric plexus was surgically lesioned and were significantly inhibited by hexamethonium. The muscarinic antagonist 4‐DAMP, which inhibits vasodilatations evoked by submucosal cholinergic vasodilator neurons, blocked dilatations elicited by mucosal stimulation and balloon distension. Maximal dilatations evoked with either sensory modality could be further enhanced when stimulated with the second modality. Dilatations evoked by stimulation of the aborad segment were similar to those elicited in the orad segment. In conclusion, sensory mechanisms in the mucosa and muscularis propria activate vasodilator pathways in the myenteric plexus which project for significant distances in both ascending and descending directions before innervating submucosal arterioles. These reflexes could co‐ordinate mucosal blood flow during multiple motor events such as peristalsis and intestinal mixing between propulsive events.
A Juvenile form of Postsynaptic Hippocampal Long‐Term Potentiation in Mice Deficient for the AMPA Receptor Subunit GluR‐A
Authors: Jensen Vidar , Kaiser Katharina M. M. , Borchardt Thilo , Adelmann Giselind , Rozov Andrei , Burnashev Nail , Brix Christian , Frotscher Michael , Andersen Per , Hvalby Øivind , Sakmann Bert , Seeburg Peter H. , Sprengel Rolf
Published: October 2003

In adult mice, long‐term potentiation (LTP) of synaptic transmission at CA3‐to‐CA1 synapses induced by tetanic stimulation requires l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors containing GluR‐A subunits. Here, we report a GluR‐A‐independent form of LTP, which is comparable in size to LTP in wild‐type mice at postnatal day 14 (P14) but diminishes between P14 and P42 in brain slices of GluR‐A‐deficient mice. The GluR‐A‐independent form of LTP is sensitive to d(−)‐2‐amino‐5‐phosphonopentanoic acid (d‐AP5), but lacks short‐term potentiation (STP) and can also be observed in the pairing induction protocol. As judged by unaltered paired‐pulse facilitation, this LTP form is postsynaptically expressed despite depleted extrasynaptic AMPA receptor pools with reduced levels of GluR‐B, which accumulates in somata and synapses of CA1 pyramidal neurons in GluR‐A‐deficient mice. Our results show that in the developing hippocampus synaptic plasticity can be expressed by AMPA receptors lacking the GluR‐A subunit.
Buffer Kinetics Shape the Spatiotemporal Patterns of IP3‐Evoked Ca2+ Signals
Authors: Dargan Sheila L. , Parker Ian
Published: October 2003

Ca2+ liberation through inositol 1,4,5‐trisphosphate receptors (IP3Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca2+ signals. IP3Rs display Ca2+‐induced Ca2+ release (CICR), but are grouped in clusters so that regenerative Ca2+ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca2+ diffusion and CICR. We used confocal microscopy and photoreleased IP3 in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca2+ buffers. EGTA (a buffer with slow ‘on‐rate’) speeded Ca2+ signals and ‘balkanized’ Ca2+ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca2+ responses and promoted ‘globalization’ of spatially uniform Ca2+ signals. These actions are likely to arise through differential effects on Ca2+ feedback within and between IP3R clusters, because Ca2+ signals evoked by influx through voltage‐gated channels were little affected. We propose that cell‐specific expression of Ca2+‐binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3‐evoked Ca2+ signals for specific physiological roles.
Purification and Characterization of a Novel Bacteriocin Produced by
Enterococcus faecalis
Strain RJ-11
Authors: Yamamoto Yukio , Togawa Yoshikazu , Shimosaka Makoto , Okazaki Mitsuo
Published: October 2003

Lactic acid bacteria exhibiting activity against the gram-positive bacterium
Bacillus subtilis
were isolated from rice bran. One of the isolates, identified as
Enterococcus faecalis
RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by
Enterococcus faecium
.
Glutathione Protects
Lactococcus lactis
against Oxidative Stress
Authors: Li Yin , Hugenholtz Jeroen , Abee Tjakko , Molenaar Douwe
Published: October 2003

Glutathione was found in several dairy
Lactococcus lactis
strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium,
L. lactis
subsp.
cremoris
strain SK11 was able to accumulate up to ∼60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H
2
O
2
compared to the resistance of cells without intracellular glutathione. The resistance to H
2
O
2
treatment was found to be dependent on the accumulation of glutathione in 16 strains of
L. lactis
tested. We propose that by taking up glutathione,
L. lactis
might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H
2
O
2
. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of
L. lactis
, but the activities of different strains were very variable. In general, the glutathione reductase activities of
L. lactis
subsp.
lactis
are higher than those of
L. lactis
subsp.
cremoris
, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in
L. lactis
could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.
Diversity of Nitrile Hydratase and Amidase Enzyme Genes in
Rhodococcus erythropolis
Recovered from Geographically Distinct Habitats
Authors: Brandão Pedro F. B. , Clapp Justin P. , Bull Alan T.
Published: October 2003

A molecular screening approach was developed in order to amplify the genomic region that codes for the α- and β-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as
Rhodococcus erythropolis
. A hydratase PCR product was also obtained from
R. erythropolis
DSM 43066
T
, which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the
R. erythropolis
strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the
R. erythropolis
strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel
R. erythropolis
enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in
R. erythropolis
. Our findings indicate that the NHase and amidase genes present in geographically distinct
R. erythropolis
strains are not globally mixed.
Development of an Improved Selective Agar Medium for Isolation of
Yersinia pestis
Authors: Ber Raphael , Mamroud Emanuelle , Aftalion Moshe , Tidhar Avital , Gur David , Flashner Yehuda , Cohen Sara
Published: October 2003

Existing media designed for selective isolation of clinically important members of the genus
Yersinia
were found to be unsatisfactory for the growth and isolation of
Yersinia pestis
. We report the development of a new selective agar medium (termed BIN) that supports the growth of
Y. pestis
. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of
Y. pestis
EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of
Y. pestis
than are the conventional semisolid media MacConkey agar and
Yersinia
-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent
Y. pestis
from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of
Y. pestis
from various backgrounds.
Conditional Survival as a Selection Strategy To Identify Plant-Inducible Genes of
Pseudomonas syringae
Authors: Marco Maria L. , Legac Jennifer , Lindow Steven E.
Published: October 2003

A novel strategy termed habitat-inducible rescue of survival (HIRS) was developed to identify genes of
Pseudomonas syringae
that are induced during growth on bean leaves. This strategy is based on the complementation of
metXW
, two cotranscribed genes that are necessary for methionine biosynthesis and required for survival of
P. syringae
on bean leaves exposed to conditions of low humidity. We constructed a promoter trap vector, pTrap, containing a promoterless version of the wild-type
P. syringae metXW
genes. Only with an active promoter fused to
metXW
on pTrap did this plasmid restore methionine prototrophy to the
P. syringae metXW
mutant B7MX89 and survival of this strain on bean leaves. To test this method, a partial library of
P. syringae
genomic DNA was constructed in pTrap and a total of 1,400 B7MX89 pTrap clones were subjected to HIRS selection on bean leaves. This resulted in the enrichment of five clones, each with a unique
Rsa
I restriction pattern of their DNA insert. Sequence analysis of these clones revealed those
P. syringae
genes for which putative plant-inducible activity could be assigned. Promoter activity experiments with a
gfp
reporter gene revealed that these plant-inducible gene promoters had very low levels of expression in minimal medium. Based on green fluorescent protein fluorescence levels, it appears that many
P. syringae
genes have relatively low expression levels and that the
metXW
HIRS strategy is a sensitive method to detect weakly expressed
P. syringae
genes that are active on plants. Furthermore, we found that protected sites on the leaf surface provided a higher level of enrichment for
P. syringae
expressing
metXW
than exposed sites. Thus, the
metXW
HIRS strategy should lead to the identification of
P. syringae
genes that are expressed primarily in these areas on the leaf.
Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A byClostridium beijerinckiiATCC 25752
Authors: Kemperman Robèr , Jonker Marnix , Nauta Arjen , Kuipers Oscar P. , Kok Jan
Published: October 2003

A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A ofClostridium beijerinckiiATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified. The genes are tightly organized in overlapping open reading frames. Heterologous expression of circularin A inEnterococcus faecaliswas achieved, and five genes were identified as minimally required for bacteriocin production and secretion. Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain. Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A. The fifth gene,cirE, confers immunity towards circularin A when expressed in eitherLactococcus lactisorE. faecalisand is needed in order to allow the bacteria to produce bacteriocin. Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD.
Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization
Authors: Zwart Gabriel , van Hannen Erik J. , Kamst-van Agterveld Miranda P. , Van der Gucht Katleen , Lindström Eva S. , Van Wichelen Jeroen , Lauridsen Torben , Crump Byron C. , Han Suk-Kyun , Declerck Steven
Published: October 2003

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster
Synechococcus
6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.
Engineering Redox Cofactor Regeneration for Improved Pentose Fermentation in
Saccharomyces cerevisiae
Authors: Verho Ritva , Londesborough John , Penttilä Merja , Richard Peter
Published: October 2003

Pentose fermentation to ethanol with recombinant
Saccharomyces cerevisiae
is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses
d
-xylose and
l
-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD
+
, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (
ZWF1
). To facilitate NADPH regeneration, we expressed the recently discovered gene
GDP1
, which codes for a fungal NADP
+
-dependent
d
-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an
S. cerevisiae
strain with the
d
-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO
2
production. The resulting strain fermented
d
-xylose to ethanol with a higher rate and yield than the corresponding strain without
GDP1
; i.e., the levels of the unwanted side products xylitol and CO
2
were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO
2
production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD
+
. The deletion of the gene
ZWF1
(which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of
GDP1
further stimulated
d
-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO
2
from
d
-xylose to a strain that produced mainly ethanol under anaerobic conditions.
Dual Resistance to
Bacillus thuringiensis
Cry1Ac and Cry2Aa Toxins in
Heliothis virescens
Suggests Multiple Mechanisms of Resistance
Authors: Jurat-Fuentes Juan Luis , Gould Fred L. , Adang Michael J.
Published: October 2003

One strategy for delaying evolution of resistance to
Bacillus thuringiensis
crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In
B. thuringiensis-
transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of
Heliothis virescens
(tobacco budworm). After previous laboratory selection with Cry1Ac,
H. virescens
strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in
H. virescens
, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of
H. virescens
to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.
PCR Detection and Analysis of the Free-Living Amoeba
Naegleria
in Hot Springs in Yellowstone and Grand Teton National Parks
Authors: Sheehan Kathy B. , Fagg Jennifer A. , Ferris Michael J. , Henson Joan M.
Published: October 2003

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for
Naegleria
to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen
Naegleria fowleri
.
Cloning, Characterization, and Functional Expression of the
Klebsiella oxytoca
Xylodextrin Utilization Operon (
xynTB
) in
Escherichia coli
Authors: Qian Yilei , Yomano L. P. , Preston J. F. , Aldrich H. C. , Ingram L. O.
Published: October 2003

Escherichia coli
is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from
Klebsiella oxytoca
M5A1 and are functionally expressed in ethanologenic
E. coli
. The xylosidase encoded by
xynB
contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The
xynT
gene encodes a membrane protein containing the MelB domain (COG2211) found in Na
+
/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both
K. oxytoca
and recombinant
E. coli
. Homologs of this operon were found in
Klebsiella pneumoniae
,
Lactobacillus lactis
,
E. coli
,
Clostridium acetobutylicum
, and
Bacillus subtilis
based on sequence comparisons. Based on similarities in protein sequence, the
xynTB
genes in
K. oxytoca
appear to have originated from a gram-positive ancestor related to
L. lactis
. Functional expression of
xynB
allowed ethanologenic
E. coli
to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant
E. coli
was increased when a full-length
xynT
gene was included with
xynB
, consistent with
xynT
functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant
E. coli
than
K. oxytoca
M5A1 (the source of
xynT
and
xynB
). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.
Compound-Specific Isotopic Fractionation Patterns Suggest Different Carbon Metabolisms among
Chloroflexus
-Like Bacteria in Hot-Spring Microbial Mats
Authors: van der Meer Marcel T. J. , Schouten Stefan , Sinninghe Damsté Jaap S. , de Leeuw Jan W. , Ward David M.
Published: October 2003

Stable carbon isotope fractionations between dissolved inorganic carbon and lipid biomarkers suggest photoautotrophy by
Chloroflexus
-like organisms in sulfidic and nonsulfidic Yellowstone hot springs. Where co-occurring, cyanobacteria appear to cross-feed
Chloroflexus
-like organisms supporting photoheterotrophy as well, although the relatively small
13
C fractionation associated with cyanobacterial sugar biosynthesis may sometimes obscure this process.
Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (
Cubitermes
spp.)
Authors: Schmitt-Wagner Dirk , Friedrich Michael W. , Wagner Bianca , Brune Andreas
Published: October 2003

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two
Cubitermes
species (
Termitidae: Termitinae
). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of
Cubitermes orthognathus
represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the
Cytophaga-Flexibacter-Bacteroides
group, various subgroups of
Proteobacteria
, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from
C. orthognathus
. The abundance and axial distribution of major phylogenetic groups in the gut of
Cubitermes ugandensis
were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the
Planctomycetales
revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from
C. orthognathus
indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different
Cubitermes
species.
Axial Dynamics, Stability, and Interspecies Similarity of Bacterial Community Structure in the Highly Compartmentalized Gut of Soil-Feeding Termites (
Cubitermes
spp.)
Authors: Schmitt-Wagner Dirk , Friedrich Michael W. , Wagner Bianca , Brune Andreas
Published: October 2003

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of
Cubitermes
spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in
Cubitermes orthognathus
confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different
Cubitermes
species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.
Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation
Authors: Varela Elisa , Tien Ming
Published: October 2003

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe
2+
and H
2
O
2
in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe
3+
reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe
3+
at a rate constant of 4.5 × 10
3
M
−1
s
−1
at pH 4.0. Fe
2+
is also very stable at a low pH. H
2
O
2
generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe
3+
. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe
3+
. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 μM), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.
IS
Lpl1
Is a Functional IS
30
-Related Insertion Element in
Lactobacillus plantarum
That Is Also Found in Other Lactic Acid Bacteria
Authors: Nicoloff Hervé , Bringel Françoise
Published: October 2003

We describe the first functional insertion sequence (IS) element in
Lactobacillus plantarum
. IS
Lpl1
, an IS
30
-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene
upp
on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: IS
Lpl2
, an IS
150
-related element adjacent to IS
Lpl1
. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of IS
Lpl1
-related sequences in different
L. plantarum
strains as well as in
Pediococcus
strains. The IS
Lpl1
pattern changed over many generations of the strain
L. plantarum
NCIMB 1406. This finding strongly supports our hypothesis that IS
Lpl1
is a mobile element in
L. plantarum
. Database analysis revealed five quasi-identical IS
Lpl1
elements in
Lactobacillus
,
Pediococcus
, and
Oenococcus
strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, IS
Lpl1
was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. IS
Lpl1
is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.
Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge
Authors: Malik Anushree , Sakamoto Masashi , Hanazaki Shohei , Osawa Masamitsu , Suzuki Takanori , Tochigi Masaki , Kakii Kazuo
Published: October 2003

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with
Acinetobacter johnsonii
S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with
Acinetobacter junii
S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes.
A. johnsonii
S35 could coaggregate with strains of several genera, such as
Oligotropha carboxidovorans
,
Microbacterium esteraromaticum
, and
Xanthomonas
spp. The role of
Acinetobacter
isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.
Molecular Diversity of Sulfate-Reducing Bacteria from Two Different Continental Margin Habitats
Authors: Liu Xueduan , Bagwell Christopher E. , Wu Liyou , Devol Allan H. , Zhou Jizhong
Published: October 2003

This study examined the natural diversity and distributions of sulfate-reducing bacteria along a natural carbon gradient extending down the shelf-slope transition zone of the eastern Pacific continental margin. Dissimilatory (bi)sulfite reductase gene sequences (
dsrAB
) were PCR amplified and cloned from five different sampling sites, each at a discrete depth, from two different margin systems, one off the Pacific coast of Mexico and another off the coast of Washington State. A total of 1,762 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis. The majority of the gene sequences recovered showed site and depth restricted distributions; however, a limited number of gene sequences were widely distributed within and between the margin systems. Cluster analysis identified 175 unique RFLP patterns, and nucleotide sequences were determined for corresponding clones. Several different continental margin DsrA sequences clustered with those from formally characterized taxa belonging to the delta subdivision of the class
Proteobacteria
(
Desulfobulbus propionicus
,
Desulfosarcina variabilis
) and the
Bacillus
-
Clostridium
(
Desulfotomaculum putei
) divisions, although the majority of the recovered sequences were phylogenetically divergent relative to all of the other DsrA sequences available for comparison. This study revealed extensive new genetic diversity among sulfate-reducing bacteria in continental margin sedimentary habitats, which appears to be tightly coupled to slope depth, specifically carbon bioavailability.
RpoS-Dependent Stress Response and Exoenzyme Production in
Vibrio vulnificus
Authors: Hülsmann A. , Rosche T. M. , Kong I.-S. , Hassan H. M. , Beam D. M. , Oliver J. D.
Published: October 2003

Vibrio vulnificus
is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens,
V. vulnificus
must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of
V. vulnificus
to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An
rpoS
mutant of
V. vulnificus
strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the
rpoS
mutant was severely diminished. Overall, these studies suggest that
rpoS
in
V. vulnificus
is important for adaptation to environmental changes and may have a role in virulence.
Patterns of Community Change among Ammonia Oxidizers in Meadow Soils upon Long-Term Incubation at Different Temperatures
Authors: Avrahami Sharon , Conrad Ralf
Published: October 2003

The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils. Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10°C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22°C). The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37°C). OMS soil was also incubated with less fertilizer than the other soils. The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of theamoAgene, which codes for the α subunit of ammonia monooxygenase. AllamoAgene sequences found belonged to the genusNitrosospira. The analysis showed community change due to temperature both in moist soil and in the soil slurry. Two patterns of community change were observed. One pattern was a change between the differentNitrosospiraclusters, which was observed in moist soil and slurry incubations of GMS and OMS.NitrosospiraAmoA cluster 1 was mainly detected below 30°C, whileNitrosospiracluster 4 was predominant at 25°C.Nitrosospiraclusters 3a, 3b, and 9 dominated at 30°C. The second pattern, observed in KMS, showed a community shift predominantly within a singleNitrosospiracluster. The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively toNitrosospiracluster 3a. We conclude that ammonia oxidizer populations are influenced by temperature. In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers. Thus,Nitrosospiracluster 1 was absent in OMS soil treated with less fertilizer, whileNitrosospiracluster 9 was only found in the sample given less fertilizer.
Immobilization of Arsenite and Ferric Iron by
Acidithiobacillus ferrooxidans
and Its Relevance to Acid Mine Drainage
Authors: Duquesne K. , Lebrun S. , Casiot C. , Bruneel O. , Personné J.-C. , Leblanc M. , Elbaz-Poulichet F. , Morin G. , Bonnefoy V.
Published: October 2003

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An
Acidithiobacillus ferrooxidans
strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This
A. ferrooxidans
strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the
A. ferrooxidans
species, as such a feature was observed with one private and three collection strains, one of which was the type strain.