bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTA 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.