bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bioprospecting for Trichothecene 3-
O
-Acetyltransferases in the Fungal Genus
Fusarium
Yields Functional Enzymes with Different Abilities To Modify the Mycotoxin Deoxynivalenol
Authors: Khatibi Piyum A. , Newmister Sean A. , Rayment Ivan , McCormick Susan P. , Alexander Nancy J. , Schmale David G. 
Published: December 2010

ABSTRACT

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-
O
-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of
Fusarium
(
F. graminearum
and
F. sporotrichioides
), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of
Fusarium
. We cloned
TRI101
or
TRI201
genes from seven different species of
Fusarium
and found genetic identity between sequences ranging from 66% to 98%.
In vitro
feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the
Fusarium
species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from
F. sporotrichioides
having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 10
4
M
−1
·s
−1
to 4.7 × 10
6
M
−1
·s
−1
. Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of
TRI101/TRI201
from 31 species of
Fusarium
(including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional
TRI101/TRI201
genes, some with the potential to outperform those evaluated in the present study.
High-Throughput Plasmid Content Analysis of
Borrelia burgdorferi
B31 by Using Luminex Multiplex Technology
Authors: Norris Steven J. , Howell Jerrilyn K. , Odeh Evelyn A. , Lin Tao , Gao Lihui , Edmondson Diane G. 
Published: December 2010

ABSTRACT

Borrelia burgdorferi
, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during
in vitro
culture. The analysis of
B. burgdorferi
plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain
B. burgdorferi
B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many
B. burgdorferi
strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage
B. burgdorferi
B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease
Borrelia
studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease
Borrelia
strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.
Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy
Authors: Dohnalkova Alice C. , Marshall Matthew J. , Arey Bruce W. , Williams Kenneth H. , Buck Edgar C. , Fredrickson James K. 
Published: December 2010

ABSTRACTMicrobe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of microscale associations. Electron microscopy has been used extensively for geomicrobial investigations, and although usedbona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions by conventional electron microscopy approaches with imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state.In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding the nature of interactions between microbial extracellular polymers and their environment.
Natural and Engineered Hydroxyectoine Production Based on the
Pseudomonas stutzeri ectABCD-ask
Gene Cluster
Authors: Seip Britta , Galinski Erwin A. , Kurz Matthias 
Published: December 2010

ABSTRACT

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster,
ectABCD-ask
, in
Pseudomonas stutzeri
DSM5190
T
and evaluate the suitability of
P. stutzeri
DSM5190
T
for hydroxyectoine production. Furthermore, we present information on heterologous
de novo
production of the compatible solute hydroxyectoine in
Escherichia coli
. In this host, the
P. stutzeri
gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in
E. coli
under the conditions employed exceeded that of the natural producer
Pseudomonas stutzeri
and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.
Possible Implications of Chytrid Parasitism for Population Subdivision in Freshwater Cyanobacteria of the Genus
Planktothrix
Authors: Sønstebø Jørn Henrik , Rohrlack Thomas 
Published: December 2010

ABSTRACT

Populations of the cyanobacterium
Planktothrix
comprise multiple coexisting oligopeptide chemotypes that can behave differently in nature. We tested whether this population subdivision can, in principle, be driven by parasitic chytrid fungi, which are almost neglected agents of
Planktothrix
mortality. Two chytrid strains, Chy-Lys2009 and Chy-Kol2008, were isolated from
Planktothrix
-dominated lakes in Norway. The two strains shared 98.2% and 86.2% of their 28S and internal transcribe spacer rRNA gene sequences, respectively. A phylogenetic analysis placed them in the order Rhizophydiales family Angulomycetaceae. Chy-Lys2009 and Chy-Kol2008 could completely lyse
Planktothrix
cultures within days, while they failed to infect other filamentous cyanobacteria. The effect on
Planktothrix
was chemotype dependent, and both chytrid strains showed distinct chemotype preferences. These findings identify chytrid fungi infecting
Planktothrix
as highly potent and specialized parasites which may exert strong selective pressure on their hosts. According to established hypotheses on host-parasite coevolution, parasitism with the above properties may result in subdivision of
Planktothrix
populations into coexisting chemotypes and periodic shifts in the relative
Planktothrix
chemotype composition. These predictions are in agreement with field observations. Moreover, a genetic analysis verified the co-occurrence of Chy-Lys2009 and Chy-Kol2008 or related chytrid strains along with distinct
Planktothrix
chemotypes in at least one water body. Our findings are consistent with a scenario where chytrid parasitism is one driving force of
Planktothrix
population subdivision, which in turn leads to polymorphism in parasitic chytrid fungi. Future studies should test the validity of this scenario under field conditions.
Inhibition of Cereulide Toxin Synthesis by Emetic
Bacillus cereus
via Long-Chain Polyphosphates
Authors: Frenzel Elrike , Letzel Thomas , Scherer Siegfried , Ehling-Schulz Monika 
Published: December 2010

ABSTRACT

Severe intoxications caused by the
Bacillus cereus
emetic toxin cereulide can hardly be prevented due to the ubiquitous distribution and heat resistance of spores and the extreme thermal and chemical stability of cereulide. It would therefore be desirable to inhibit cereulide synthesis during food manufacturing processes or in prepared foods, which are stored under time-temperature abuse conditions. Toward this end, the impacts of three long-chain polyphosphate (polyP) formulations on growth and cereulide production were examined. The inhibition was dependent on the concentration and the type of the polyP blend, indicating that polyPs and not the orthophosphates were effective. Quantitative PCR (qPCR) monitoring at sublethal concentrations revealed that polyPs reduced the transcription of
ces
nonribosomal peptide synthetase (NRPS) genes by 3- to 4-fold along with a significantly reduced toxin production level. At lower concentrations, toxin synthesis was decreased, although the growth rate was not affected. These data indicate a differential effect on toxin synthesis independent of growth inhibition. The inhibition of toxin synthesis in food was also observed. Despite the growth of
B. cereus
, toxin synthesis was reduced by 70 to 100% in two model food systems (reconstituted infant food and oat milk), which were analyzed with HEp-2 cell culture assays and high-performance liquid chromatography (HPLC)/electrospray ionization-time of flight mass spectrometry (ESI-TOF-MS). Accordingly,
ces
promoter activity was strongly downregulated, as visualized by using a
lux
-based reporter strain. These data illustrate the potential of polyphosphate formulations to reduce the risk of cereulide synthesis in food and may contribute to targeted hurdle concepts.
Metagenomic Analyses: Past and Future Trends
Authors: Simon Carola , Daniel Rolf 
Published: December 2010

ABSTRACT
Metagenomics has revolutionized microbiology by paving the way for a cultivation-independent assessment and exploitation of microbial communities present in complex ecosystems. Metagenomics comprising construction and screening of metagenomic DNA libraries has proven to be a powerful tool to isolate new enzymes and drugs of industrial importance. So far, the majority of the metagenomically exploited habitats comprised temperate environments, such as soil and marine environments. Recently, metagenomes of extreme environments have also been used as sources of novel biocatalysts. The employment of next-generation sequencing techniques for metagenomics resulted in the generation of large sequence data sets derived from various environments, such as soil, the human body, and ocean water. Analyses of these data sets opened a window into the enormous taxonomic and functional diversity of environmental microbial communities. To assess the functional dynamics of microbial communities, metatranscriptomics and metaproteomics have been developed. The combination of DNA-based, mRNA-based, and protein-based analyses of microbial communities present in different environments is a way to elucidate the compositions, functions, and interactions of microbial communities and to link these to environmental processes.
Systematic Screen of Schizosaccharomyces pombe Deletion Collection Uncovers Parallel Evolution of the Phosphate Signal Transduction Pathway in Yeasts
Authors: Henry Theresa C. , Power Juliette E. , Kerwin Christine L. , Mohammed Aishat , Weissman Jonathan S. , Cameron Dale M. , Wykoff Dennis D. 
Published: December 2010

ABSTRACT

The phosphate signal transduction (PHO) pathway, which regulates genes in response to phosphate starvation, is well defined in
Saccharomyces cerevisiae
. We asked whether the PHO pathway was the same in the distantly related fission yeast
Schizosaccharomyces pombe
. We screened a deletion collection for mutants aberrant in phosphatase activity, which is primarily a consequence of
pho1
+
transcription. We identified a novel zinc finger-containing protein (encoded by
spbc27b12.11c
+
), which we have named
pho7
+
, that is essential for
pho1
+
transcriptional induction during phosphate starvation. Few of the
S. cerevisiae
genes involved in the PHO pathway appear to be involved in the regulation of the phosphate starvation response in
S. pombe
. Only the most upstream genes in the PHO pathway in
S. cerevisiae
(
ADO1
,
DDP1
, and
PPN1
) share a similar role in both yeasts. Because
ADO1
and
DDP1
regulate ATP and IP
7
levels, we hypothesize that the ancestor of these yeasts must have sensed similar metabolites in response to phosphate starvation but have evolved distinct mechanisms in parallel to sense these metabolites and induce phosphate starvation genes.
Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena
Authors: Leondaritis George , Sarri Theoni , Dafnis Ioannis , Efstathiou Antonia , Galanopoulou Dia 
Published: December 2010

ABSTRACTEukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], produce the Ca2+-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence thatTetrahymenaspecies contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca2+. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca2+, modestly activated by GTPγSin vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLCin vivoresulted in rapid upregulation of PtdIns(4,5)P2levels, suggesting its functional importance in regulating phosphoinositide turnover inTetrahymena. Byin silicoand molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in threeT. thermophilastrains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P3-Ca2+regulatoryaxis in ciliates.
Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in
Geobacter sulfurreducens
Authors: Rollefson Janet B. , Stephen Camille S. , Tien Ming , Bond Daniel R. 
Published: December 2010

ABSTRACT

Transposon insertions in
Geobacter sulfurreducens
GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in
trans
. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-
d
-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type
G. sulfurreducens
which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of
c
-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of
c
-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the
xap
(extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the
Geobacter
outer membrane.
Identification of Missing Genes and Enzymes for Autotrophic Carbon Fixation in
Crenarchaeota
Authors: Ramos-Vera W. Hugo , Weiss Michael , Strittmatter Eric , Kockelkorn Daniel , Fuchs Georg 
Published: December 2010

ABSTRACT

Two autotrophic carbon fixation cycles have been identified in
Crenarchaeota
. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the
Thermoproteales
and
Desulfurococcales
. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic
Sulfolobales
; a similar cycle may operate in autotrophic aerobic marine
Crenarchaeota
. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of succinyl-CoA to two molecules of acetyl-CoA in
Thermoproteus neutrophilus
(
Thermoproteales
),
Ignicoccus hospitalis
(
Desulfurococcales
), and
Metallosphaera sedula
(
Sulfolobales
). The identified enzymes/genes include succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(
S
)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.
Pyruvate Formate Lyase Acts as a Formate Supplier for Metabolic Processes during Anaerobiosis in
Staphylococcus aureus
Authors: Leibig Martina , Liebeke Manuel , Mader Diana , Lalk Michael , Peschel Andreas , Götz Friedrich 
Published: December 2010

ABSTRACT

Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in
Staphylococcus aureus
biofilms. To investigate their physiological role, we constructed
fdh
and
pfl
deletion mutants (Δ
fdh
and Δ
pfl
). Although formate dehydrogenase activity in the
fdh
mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the
pfl
mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the
pfl
mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δ
pfl
mutant. In compensation reactions, serine and threonine were consumed better by the Δ
pfl
mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δ
pfl
mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and
arc
operon transcription were increased in the Δ
pfl
mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H
+
, whose regeneration requires respiration.
Unexpected Coregulator Range for the Global Regulator Lrp of
Escherichia coli
and
Proteus mirabilis
Authors: Hart Benjamin R. , Blumenthal Robert M. 
Published: December 2010

ABSTRACT

The Lrp/AsnC family of transcription factors links gene regulation to metabolism in bacteria and archaea. Members of this family, collectively, respond to a wide range of amino acids as coregulators. In
Escherichia coli
, Lrp regulates over 200 genes directly and is well known to respond to leucine and, to a somewhat lesser extent, alanine. We focused on Lrp from
Proteus mirabilis
and
E. coli
, orthologs with 98% identity overall and identical helix-turn-helix motifs, for which a previous study nevertheless found functional differences. Sequence differences between these orthologs, within and adjacent to the amino acid-responsive RAM domain, led us to test for differential sensitivity to coregulatory amino acids. In the course of this investigation, we found, via
in vivo
reporter fusion assays and
in vitro
electrophoretic mobility shift experiments, that
E. coli
Lrp itself responded to a broader range of amino acids than was previously appreciated. In particular, for both the
E. coli
and
P. mirabilis
orthologs, Lrp responsiveness to methionine was similar in magnitude to that to leucine. Both Lrp orthologs are also fairly sensitive to Ile, His, and Thr. These observations suggest that Lrp ties gene expression in the
Enterobacteriaceae
rather extensively to physiological status, as reflected in amino acid pools. These findings also have substantial implications for attempts to model regulatory architecture from transcriptome measurements or to infer such architecture from genome sequences, and they suggest that even well-studied regulators deserve ongoing exploration.