bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin
Authors: Weng Li-Xing , Wang Lian-Hui , Xu Jin-Ling , Wu Ji-En , Li Qi , Zhang Lian-Hui 
Published: March 2005

ABSTRACT

The
albA
gene of
Klebsiella oxytoca
encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10
−8
M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys
106
, Trp
110
, Tyr
113
, Leu
114
, Tyr
126
, Pro
134
, and Trp
162
with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His
125
, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.
Functional Characterization of Two Cytochrome P450 Monooxygenase Genes,
P450
-
1
and
P450
-
4
, of the Gibberellic Acid Gene Cluster in
Fusarium proliferatum
(
Gibberella fujikuroi
MP-D)
Authors: Malonek S. , Rojas M. C. , Hedden P. , Gaskin P. , Hopkins P. , Tudzynski B. 
Published: March 2005

ABSTRACT

Gibberella fujikuroi
is a species complex with at least nine different biological species, termed mating populations (MPs) A to I (MP-A to MP-I), known to produce many different secondary metabolites. So far, gibberellin (GA) production is restricted to
Fusarium fujikuroi
(
G. fujikuroi
MP-C), although at least five other MPs contain all biosynthetic genes. Here, we analyze the GA gene cluster and GA pathway in the closest related species,
Fusarium proliferatum
(MP-D), and demonstrate that the GA genes share a high degree of sequence homology with the corresponding genes of MP-C. The GA production capacity was restored after integration of the entire GA gene cluster from MP-C, indicating the existence of an active regulation system in
F. proliferatum
. The results further indicate that one reason for the loss of GA production is the accumulation of several mutations in the coding and 5′ noncoding regions of the
ent
-kaurene oxidase gene,
P450
-
4
.
“
Endomicrobia
”: Cytoplasmic Symbionts of Termite Gut Protozoa Form a Separate Phylum of Prokaryotes
Authors: Stingl Ulrich , Radek Renate , Yang Hong , Brune Andreas 
Published: March 2005

ABSTRACT

Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of
Reticulitermes santonensis
belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of
Reticulitermes speratus
that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that
Trichonympha agilis
(Hypermastigida) and
Pyrsonympha vertens
(Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 μm in length and 0.3 μm in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus “
Endomicrobium
.” Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name “
Endomicrobia
,” are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus
Cryptocercus
, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.
Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms
Authors: Scheldeman Patsy , Pil Annelies , Herman Lieve , De Vos Paul , Heyndrickx Marc 
Published: March 2005

ABSTRACTThe presence of highly heat-resistant spores ofBacillus sporothermoduransin ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera.Bacillus licheniformisandBacillus palliduswere the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.
Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production byLactococcus lactis
Authors: Wisselink H. Wouter , Moers Antoine P. H. A. , Mars Astrid E. , Hoefnagel Marcel H. N. , de Vos Willem M. , Hugenholtz Jeroen 
Published: March 2005

ABSTRACTTo achieve high mannitol production byLactococcus lactis, the mannitol 1-phosphatase gene ofEimeria tenellaand the mannitol 1-phosphate dehydrogenase genemtlDofLactobacillus plantarumwere cloned in the nisin-dependentL. lactisNICE overexpression system. As predicted by a kineticL. lactisglycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by theL. lactisparental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficientL. lactisstrain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.
Intracellular pH Distribution in
Saccharomyces cerevisiae
Cell Populations, Analyzed by Flow Cytometry
Authors: Valli Minoska , Sauer Michael , Branduardi Paola , Borth Nicole , Porro Danilo , Mattanovich Diethard 
Published: March 2005

ABSTRACT

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of
Saccharomyces cerevisiae
cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on
S. cerevisiae
cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.
Two α(1-3) Glucan Synthases with Different Functions in
Aspergillus fumigatus
Authors: Beauvais A. , Maubon D. , Park S. , Morelle W. , Tanguy M. , Huerre M. , Perlin D. S. , Latgé J. P. 
Published: March 2005

ABSTRACT

α(1-3) glucan is a main component of the
Aspergillus fumigatus
cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in
A. fumigatus
,
AGS1
and
AGS2
, are highly homologous to the
AGS
genes of
Schizosaccharomyces pombe
, which encode putative α(1-3) glucan synthases. The predicted Ags proteins of
A. fumigatus
have an estimated molecular mass of 270 kDa.
AGS1
and
AGS2
were disrupted in
A. fumigatus
. Both Δags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Δags1 presented a reduction in the α(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of
A. fumigatus
mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Δags1 and Δags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall α(1-3) glucan does not play a significant role in
A. fumigatus
pathogenicity.
Role of
cis
-Acting Sites
NorL
, a TATA Box, and
AflR1
in
nor-1
Transcriptional Activation in
Aspergillus parasiticus
Authors: Miller Michael J. , Roze Ludmila V. , Trail Frances , Linz John E. 
Published: March 2005

ABSTRACT

The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus
Aspergillus. nor-1
encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (
AflR1
). Proteins in
Aspergillus parasiticus
cell extracts and AflR expressed in
Escherichia coli
do not bind to
A. parasiticus AflR1
in vitro, so it was not clear if this site was required for
nor-1
expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in
nor-1
expression in
A. parasiticus
and identified additional
cis
-acting sites required for maximum
nor-1
transcriptional activation. Deletion and substitution of
AflR1
in the
nor-1
promoter in
A. parasiticus nor-1
::GUS reporter strains showed that this site is required for
nor-1
transcriptional activation in vivo. Substitution of a putative TATA box in the
nor-1
promoter resulted in nondetectable β-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel
cis
-acting site, designated
NorL
, between residues −210 and −238 that was required for maximum
nor-1
transcriptional activation in
A. parasiticus
grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific
NorL
-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the
NorL
site appears only once in the aflatoxin gene cluster, its association with the
nor-1
promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.
Coexistence of Bacterial Sulfide Oxidizers, Sulfate Reducers, and Spirochetes in a Gutless Worm (Oligochaeta) from the Peru Margin
Authors: Blazejak Anna , Erséus Christer , Amann Rudolf , Dubilier Nicole 
Published: March 2005

ABSTRACTOlavius crassitunicatusis a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distantO. crassitunicatuspopulations (separated by 385 km) on the Peru margin (water depth, ∼300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the γ-Proteobacteria(Gamma 1 and 2 symbionts), two members of the δ-Proteobacteria(Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the δ-Proteobacteria(Delta 3 symbiont) was found in only one of the two host populations. Three of theO. crassitunicatusbacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts ofOlavius algarvensis,Olavius loisae, andInanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts ofO. algarvensis, and the spirochete is closely related to spirochetal symbionts ofO. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.
Natural Dissemination of
Bacillus anthracis
Spores in Northern Canada
Authors: Dragon D. C. , Bader D. E. , Mitchell J. , Woollen N. 
Published: March 2005

ABSTRACT

Soil samples were collected from around fresh and year-old bison carcasses and areas not associated with known carcasses in Wood Buffalo National Park during an active anthrax outbreak in the summer of 2001. Sample selection with a grid provided the most complete coverage of a site. Soil samples were screened for viable
Bacillus anthracis
spores via selective culture, phenotypic analysis, and PCR.
Bacillus anthracis
spores were isolated from 28.4% of the samples. The highest concentrations of
B. anthracis
spores were found directly adjacent to fresh carcasses and invariably corresponded to locations where the soil had been saturated with body fluids escaping the carcass through either natural body orifices or holes torn by scavengers. The majority of positive samples were found within 2 m of both year-old and fresh carcasses and probably originated from scavengers churning up and spreading the body fluid-saturated soil as they fed. Trails of lesser contamination radiating from the carcasses probably resulted from spore dissemination through adhesion to scavengers and through larger scavengers dragging away disarticulated limbs. Comparison of samples from minimally scavenged and fully necropsied carcass sites revealed no statistically significant difference in the level of
B. anthracis
spore contamination. Therefore, the immediate area around a suspected anthrax carcass should be considered substantially contaminated regardless of the condition of the carcass.
Chemotaxonomic Identification of Single Bacteria by Micro-Raman Spectroscopy: Application to Clean-Room-Relevant Biological Contaminations
Authors: Rösch Petra , Harz Michaela , Schmitt Michael , Peschke Klaus-Dieter , Ronneberger Olaf , Burkhardt Hans , Motzkus Hans-Walter , Lankers Markus , Hofer Stefan , Thiele Hans , Popp Jürgen 
Published: March 2005

ABSTRACTMicroorganisms, such as bacteria, which might be present as contamination inside an industrial food or pharmaceutical clean room process need to be identified on short time scales in order to minimize possible health hazards as well as production downtimes causing financial deficits. Here we describe the first results of single-particle micro-Raman measurements in combination with a classification method, the so-called support vector machine technique, allowing for a fast, reliable, and nondestructive online identification method for single bacteria.
Environmental DNA Fragment Conferring Early and Increased Sporulation and Antibiotic Production in
Streptomyces
Species
Authors: Martinez Asuncion , Kolvek Steven J. , Hopke Joern , Tiong Yip Choi Lai , Osburne Marcia S. 
Published: March 2005

ABSTRACT

Here we describe the
rep
gene, isolated from an environmental DNA library, which when transformed into
Streptomyces
species resulted in increased production of secondary metabolites and accelerated sporulation. We show that
Streptomyces lividans
strains bearing
rep
are particularly useful as expression hosts for heterologous antibiotic production.
Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from
Neurospora crassa
Authors: Woodyer Ryan , Simurdiak Michael , van der Donk Wilfred A. , Zhao Huimin 
Published: March 2005

ABSTRACT

A xylose reductase (XR) gene was identified from the
Neurospora crassa
whole-genome sequence, expressed heterologously in
Escherichia coli
, and purified as a His
6
-tagged fusion in high yield. This enzyme is one of the most active XRs thus far characterized and may be used for the in vitro production of xylitol.
Comparison of Diversities ofEscherichia coliO157 Shed from a Cohort of Spring-Born Beef Calves at Pasture and in Housing
Authors: Vali Leila , Pearce Michael C. , Wisely Karen A. , Hamouda Ahmed , Knight Hazel I. , Smith Alastair W. , Amyes Sebastian G. B. 
Published: March 2005

ABSTRACTA cohort of spring-born beef calves demonstrated limited genetic and phenotypic diversity ofEscherichia coliO157 when kept in a state of isolation. Despite this, there was a difference in the pulsed-field gel electrophoresis and phage types of isolates shed by cattle at pasture compared with those shed by the same cattle when weaned and housed.
Effect of Batch-Process Solar Disinfection on Survival of
Cryptosporidium parvum
Oocysts in Drinking Water
Authors: Méndez-Hermida F. , Castro-Hermida J. A. , Ares-Mazás E. , Kehoe S. C. , McGuigan K. G. 
Published: March 2005

ABSTRACT

The results of batch-process solar disinfection (SODIS) of
Cryptosporidium parvum
oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m
−2
) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.
Genetic Diversity in the Lactose Operons of
Lactobacillus helveticus
Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures
Authors: Callanan M. J. , Beresford T. P. , Ross R. P. 
Published: March 2005

ABSTRACT

Two novel insertion sequence elements, ISLhe
1
and ISLhe
15
, were located upstream of the genes encoding the β-galactosidase enzyme in
Lactobacillus helveticus
commercial starter strains. Strains with the IS
982
family element, ISLhe
1
, demonstrated reduced β-galactosidase activity compared to the
L. helveticus
type strain, whereas strains with the ISLhe
15
element expressed β-galactosidase in the absence of lactose.
Probiotic Bacteria May Become Dormant during Storage
Authors: Lahtinen Sampo J. , Gueimonde Miguel , Ouwehand Arthur C. , Reinikainen Johanna P. , Salminen Seppo J. 
Published: March 2005

ABSTRACT

The determination of bacterial viability in probiotic products is of economic, technological, and clinical significance. We compared four methods to enumerate three
Bifidobacterium
strains in fermented oat products during storage. A subpopulation of nonculturable cells retained a functional cell membrane typical of viable cells, indicating that probiotic bacteria become dormant during storage.
Application of a Newly Developed ARB Software-Integrated Tool for In Silico Terminal Restriction Fragment Length Polymorphism Analysis Reveals the Dominance of a Novel
pmoA
Cluster in a Forest Soil
Authors: Ricke Peter , Kolb Steffen , Braker Gesche 
Published: March 2005

ABSTRACT

TRF-CUT, an ARB-implemented tool, was developed to predict in silico the terminal restriction fragments of aligned small-subunit rRNA gene or functional gene sequences. Application of this new tool to perform directed terminal restriction fragment length polymorphism analysis of
pmoA
products obtained from a forest soil revealed that novel cluster I methanotrophic bacteria were dominant.