bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-
cis,cis
-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A
Authors: Kaulmann Ursula , Kaschabek Stefan R. , Schlömann Michael 
Published: July 2002

ABSTRACT

Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-
cis,cis
-muconate to
cis
-dienelactone (
cis
-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-
cis,cis
-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation of
cis
-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of
Pseudomonas putida
muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such as
cis,cis
-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-
cis,cis
-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-
cis,cis
-muconate. Interestingly,
cis
-dienelactone and 2-chloro-
cis
-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.
Regulation and Mechanism of Action of the Small Heat Shock Protein from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Laksanalamai Pongpan , Maeder Dennis L. , Robb Frank T. 
Published: July 2002

ABSTRACT

The small heat shock protein (sHSP) from the hyperthermophile
Pyrococcus furiosus
was specifically induced at the level of transcription by heat shock at 105°C. The gene encoding this protein was cloned and overexpressed in
Escherichia coli
. The recombinant sHSP prevented the majority of
E. coli
proteins from aggregating in vitro for up to 40 min at 105°C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56°C. Survivability of
E. coli
overexpressing the sHSP was enhanced approximately sixfold during exposure to 50°C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.
GerN, an Antiporter Homologue Important in Germination of
Bacillus cereus
Endospores
Authors: Thackray Penny D. , Behravan Javad , Southworth Thomas W. , Moir Anne 
Published: July 2002

ABSTRACT

A homologue of the
grmA
spore germination gene of
Bacillus megaterium
and of a NaH-antiporter gene (
napA
) of
Enterococcus hirae
has been identified in
Bacillus cereus
569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this
B. cereus
gene, named
gerN
, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to
l
-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 μM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 μM
l
-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of
l
-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.
YkdA and YvtA, HtrA-Like Serine Proteases in
Bacillus subtilis
, Engage in Negative Autoregulation and Reciprocal Cross-Regulation of
ykdA
and
yvtA
Gene Expression
Authors: Noone David , Howell Alistair , Collery Ross , Devine Kevin M. 
Published: July 2002

ABSTRACT

HtrA-type serine proteases participate in folding and degradation of aberrant proteins and in processing and maturation of native proteins. Mutation of the corresponding genes often confers a pleiotropic phenotype that can include temperature sensitivity, sensitivity to osmotic and oxidative stress, and attenuated virulence. There are three HtrA-type serine proteases, YkdA, YvtA, and YycK, encoded in the
Bacillus subtilis
genome. In this report we show that YkdA and YvtA display many similarities: their expression profiles during the growth cycle in wild-type and mutant backgrounds are very alike, with expression being directed by very similar promoters. Both are induced by temperature upshift and by heterologous amylases at the transition phase of the growth cycle. These characteristics are quite different for YycK, suggesting that it has a cellular function distinct from that of the other two proteases or that it performs the same function but under different conditions. We also show that inactivation of either
ykdA
or
yvtA
results in compensating overexpression of the other gene, especially during stress conditions, with a concomitant increase in resistance to heat and hydrogen peroxide stresses. Mutation of both
ykdA
and
yvtA
leads to growth defects and to thermosensitivity. The fact that their expression increases dramatically at the transition phase of the growth cycle under certain conditions suggests that the YkdA and YvtA proteases may function in the processing, maturation, or secretion of extracellular enzymes in
B. subtilis
.
Bacillus subtilis
Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture
Authors: Dauner Michael , Storni Tazio , Sauer Uwe 
Published: July 2002

ABSTRACT

The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing
Bacillus subtilis
. Based on [U-
13
C
6
]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited
B. subtilis
is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited
B. subtilis
, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.
NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of
Ralstonia eutropha
Strain Bo on Tetrahydrofurfuryl Alcohol
Authors: Schräder Thomas , Zarnt Grit , Andreesen Jan R. 
Published: July 2002

ABSTRACT

Different aldehyde dehydrogenases (AlDHs) were formed during growth of
Ralstonia eutropha
Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4.
Sugar Transport through Maltoporin of
Escherichia coli
: Role of the Greasy Slide
Authors: Van Gelder Patrick , Dumas Fabrice , Bartoldus Ingrid , Saint Nathalie , Prilipov Alexei , Winterhalter Mathias , Wang Yanfei , Philippsen Ansgar , Rosenbusch Jürg P. , Schirmer Tilman 
Published: July 2002

ABSTRACT
The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.
Glycerol-3-Phosphate-Induced Catabolite Repression inEscherichia coli
Authors: Eppler Tanja , Postma Pieter , Schütz Alexandra , Völker Uwe , Boos Winfried 
Published: July 2002

ABSTRACTThe formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction inmalTexpression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlcphosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlcdid not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlcis controlled by G3P and other phosphorylated sugars such asd-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), andd-tagatose-1,6-bisphosphate aldolase.
Acinetobacter baumannii
at a Tertiary-Care Teaching Hospital in Jerusalem, Israel
Authors: Simhon A. , Rahav G. , Shazberg G. , Block C. , Bercovier H. , Shapiro M. 
Published: July 2002

ABSTRACT

In a retrospective 10-year analysis of 3,536 patient-unique isolates,
Acinetobacter baumannii
imipenem susceptibility declined from 98.1 (1990) to 64.1% (2000), and ciprofloxacin susceptibility decreased from 50.5 to 13.1%. Imipenem median zone diameters decreased from 27.7 (1997) to 18.8 mm (2000). No outbreaks were detected. Two clusters were identified for 41 strains genotyped by pulsed-field gel electrophoresis, but imipenem resistance was not clonal.
Analysis of Bacterial Vaginosis-Related Amines in Vaginal Fluid by Gas Chromatography and Mass Spectrometry
Authors: Wolrath Helen , Forsum Urban , Larsson P. G. , Borén Hans 
Published: July 2002

ABSTRACT
The presence of various amines in vaginal fluid from women with malodorous vaginal discharge has been reported before. The investigations have used several techniques to identify the amines. However, an optimized quantification, together with a sensitive analysis method in connection with a diagnostic procedure for vaginal discharge, including the syndrome of bacterial vaginosis, as defined by the accepted “gold standard,” has not been done before. We now report a sensitive gas chromatographic and mass spectrometric method for identifying the amines isobutylamine, phenethylamine, putrescine, cadaverine, and tyramine in vaginal fluid. We used weighted samples of vaginal fluid to obtain a correct quantification. In addition, a proper diagnosis was obtained using Gram-stained smears of the vaginal fluid that were Nugent scored according to the method of Nugent et al. (R. P. Nugent et al., J. Clin. Microbiol., 29:297–301, 1991). We found that putrescine, cadaverine, and tyramine occurred in high concentrations in vaginal fluid from 24 women with Nugent scores between 7 and 10. These amines either were not found or were found only in very low concentrations in vaginal fluid from women with Nugent scores of 0 to 3. There is a strong correlation between bacterial vaginosis and the presence of putrescine, cadaverine, and tyramine in high concentrations in vaginal fluid.
PCR-Based Detection and Identification of
Burkholderia
cepacia
Complex Pathogens in Sputum from Cystic Fibrosis Patients
Authors: McDowell Andrew , Mahenthiralingam Eshwar , Moore John E. , Dunbar Kerstin E. A. , Webb A. Kevin , Dodd Mary E. , Martin S. Lorraine , Millar B. Cherie , Scott Christopher J. , Crowe Mary , Elborn J. Stuart 
Published: July 2002

ABSTRACT

PCR amplification of the
recA
gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification of
Burkholderia
cepacia
complex genomovars directly from sputum. Successful amplification of the
B
.
cepacia
complex
recA
gene from cystic fibrosis (CF) patient sputum samples containing
B
.
cepacia
genomovar I,
Burkholderia
multivorans
,
B
.
cepacia
genomovar III,
Burkholderia
stabilis
, and
Burkholderia
vietnamiensis
was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed that
recA
-based PCR could reliably detect
B
.
cepacia
complex organisms to concentrations of 10
6
CFU g of sputum
−1
. To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for
B
.
cepacia
complex infection by selective culturing and
recA
-based PCR. Selective culturing identified 19 samples with presumptive
B
.
cepacia
complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by
recA
-based PCR, while 2 samples were negative. The isolates cultured from both
recA
-negative sputum samples were subsequently identified as
Burkholderia
gladioli
. RFLP analysis of the
recA
amplicons revealed 2 patients (12%) infected with
B
.
multivorans
, 11 patients (65%) infected with
B
.
cepacia
genomovar III-A, and 4 patients (23%) infected with
B
.
cepacia
genomovar III-B. These results demonstrate the potential of
recA
-based PCR-RFLP analysis for the rapid detection and identification of
B
.
cepacia
complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by
recA
-based analysis of the isolate.
Identification of
Pandoraea
Species by 16S Ribosomal DNA-Based PCR Assays
Authors: Coenye Tom , Liu Lixia , Vandamme Peter , LiPuma John J. 
Published: July 2002

ABSTRACT

The recently described genus
Pandoraea
contains five named species (
Pandoraea apista
,
Pandoraea pulmonicola
,
Pandoraea pnomenusa
,
Pandoraea sputorum
, and
Pandoraea norimbergensis
) and four unnamed genomospecies.
Pandoraea
spp. have mainly been recovered from the respiratory tracts of cystic fibrosis (CF) patients. Accurate genus- and species-level identification by routine clinical microbiology methods is difficult, and differentiation from
Burkholderia cepacia
complex organisms may be especially problematic. This can have important consequences for the management of CF patients. On the basis of 16S ribosomal DNA sequences, PCR assays for the identification of
Pandoraea
spp. were developed. A first PCR assay was developed for the identification of
Pandoraea
isolates to the genus level. PCR assays for the identification of
P. apista
and
P. pulmonicola
as a group,
P. pnomenusa
,
P. sputorum
, and
P. norimbergensis
were also developed. All five assays were evaluated with a panel of 123 bacterial isolates that included 69
Pandoraea
sp. strains, 24
B. cepacia
complex strains, 6
Burkholderia gladioli
strains, 9
Ralstonia
sp. strains, 5
Alcaligenes xylosoxidans
strains, 5
Stenotrophomonas maltophilia
strains, and 5
Pseudomonas aeruginosa
strains. The use of these PCR assays facilitates the identification of
Pandoraea
spp. and avoids the misidentification of a
Pandoraea
sp. as a
B. cepacia
complex isolate.
Zoonotic Potential of
Enterocytozoon bieneusi
Authors: Dengjel Bianca , Zahler Monika , Hermanns Walter , Heinritzi Karl , Spillmann Thomas , Thomschke Angelika , Löscher Thomas , Gothe Rainer , Rinder Heinz 
Published: July 2002

ABSTRACT

The reservoirs and the modes of transmission of the most frequent microsporidial species in humans,
Enterocytozoon bieneusi
, are still unknown. We have examined fecal samples of 26 humans and 350 animals from 37 species to find 18 samples containing this parasite from humans, cats, pigs, cattle, and a llama. Genotypic characterization of the internal transcribed spacer of the rRNA gene resulted in 14 different genotypes, 6 of them previously undescribed. Phylogenetic analysis revealed the lack of a transmission barrier between
E. bieneusi
from humans and animals (cats, pigs, and cattle). Thus,
E. bieneusi
appears to be a zoonotic pathogen.
In Vitro Fungicidal Activities of Voriconazole, Itraconazole, and Amphotericin B against Opportunistic Moniliaceous and Dematiaceous Fungi
Authors: Espinel-Ingroff Ana 
Published: July 2002

ABSTRACT

The NCCLS proposed standard M38-P describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds); however, standard conditions are not available for testing their fungicidal activities (minimum fungicidal or lethal concentrations [MFCs]). This study evaluated the in vitro fungistatic and fungicidal activities of voriconazole, itraconazole, and amphotericin B against 260 common and emerging molds (174
Aspergillus
sp. isolates [five species], 23
Fusarium
sp. isolates [three species], 6
Paecilomyces lilacinus
isolates, 6
Rhizopus arrhizus
isolates, 23
Scedosporium
sp. isolates, 23 dematiaceous fungi, and 5
Trichoderma longibrachiatum
isolates). MICs were determined by following the NCCLS M38-P broth microdilution method. MFCs were the lowest drug dilutions that resulted in fewer than three colonies. Voriconazole showed similar or better fungicidal activity (MFC at which 90% of isolates tested are killed [MFC
90
], 1 to 2 μg/ml) than the reference agents for
Aspergillus
spp. with the exception of
Aspergillus terreus
(MFC
90
of voriconazole and amphotericin B, >8 μg/ml). The voriconazole geometric mean (G mean) MFC for
Scedosporium apiospermum
was lower (2.52 μg/ml) than those of the other two agents (5.75 to 7.5 μg/ml). In contrast, amphotericin B and itraconazole G mean MFCs for
R. arrhizus
were 2.1 to 2.2 μg/ml, but that for voriconazole was >8 μg/ml. Little or no fungicidal activity was shown for
Fusarium
spp. (2 to >8 μg/ml) and
Scedosporium prolificans
(>8 μg/ml) by the three agents, but voriconazole had some activity against
P. lilacinus
and
T. longibrachiatum
(G mean MFCs, 1.8 and 4 μg/ml, respectively). The fungicidal activity of the three agents was similar (G mean MFC, 1.83 to 2.36 μg/ml) for the dematiaceous fungi with the exception of the azole MFCs (>8 μg/ml) for some
Bipolaris spicifera
and
Dactylaria constricta
var.
gallopava
. These data extend and corroborate the available fungicidal results for the three agents. The role of the MFC as a predictor of clinical outcome needs to be established in clinical trials by following standardized testing conditions for determination of these in vitro values.
Genotypic Diversity of Mutans Streptococci in Brazilian Nursery Children Suggests Horizontal Transmission
Authors: Mattos-Graner Renata O. , Li Yihong , Caufield Page W. , Duncan Margaret , Smith Daniel J. 
Published: July 2002

ABSTRACT

Streptococcus mutans
strains were isolated from cohorts of Brazilian nursery school children and genotyped by arbitrarily primed PCR and restriction fragment length polymorphism analysis. Of 24 children with two to five
S. mutans
isolates, 29% carried two or more genotypes. The presence of matching genotypes of
S. mutans
among children attending one nursery suggests horizontal transmission.
Accurate, Noninvasive Detection of
Helicobacter pylori
DNA from Stool Samples: Potential Usefulness for Monitoring Treatment
Authors: Shuber Anthony P. , Ascaño Jennifer J. , Boynton Kevin A. , Mitchell Anastasia , Frierson Henry F. , El-Rifai Wa’el , Powell Steven M. 
Published: July 2002

ABSTRACT

A novel DNA assay demonstrating sensitive and accurate detection of
Helicobacter pylori
from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of
H. pylori
DNA during treatment. Thus, this noninvasive molecular biology-based assay has the potential to be a powerful diagnostic tool given its ability to specifically identify
H. pylori
DNA.