bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Inducible Propane Monooxygenase Is Responsible for
N
-Nitrosodimethylamine Degradation by
Rhodococcus
sp. Strain RHA1
Authors: Sharp Jonathan O. , Sales Christopher M. , LeBlanc Justin C. , Liu Jie , Wood Thomas K. , Eltis Lindsay D. , Mohn William W. , Alvarez-Cohen Lisa 
Published: September 2007

ABSTRACT

Rhodococci are common soil heterotrophs that possess diverse functional enzymatic activities with economic and ecological significance. In this study, the correlation between gene expression and biological removal of the water contaminant
N
-nitrosodimethylamine (NDMA) is explored. NDMA is a hydrophilic, potent carcinogen that has gained recent notoriety due to its environmental persistence and emergence as a widespread micropollutant in the subsurface environment. In this study, we demonstrate that
Rhodococcus
sp. strain RHA1 can constitutively degrade NDMA and that activity toward this compound is enhanced by approximately 500-fold after growth on propane. Transcriptomic analysis of RHA1 and reverse transcriptase quantitative PCR assays demonstrate that growth on propane elicits the upregulation of gene clusters associated with (i) the oxidation of propane and (ii) the oxidation of substituted benzenes. Deletion mutagenesis of
prmA
, the gene encoding the large hydroxylase component of propane monooxygenase, abolished both growth on propane and removal of NDMA. These results demonstrate that propane monooxygenase is responsible for NDMA degradation by RHA1 and explain the enhanced cometabolic degradation of NDMA in the presence of propane.
Genetic Method To Analyze Essential Genes of
Escherichia coli
Authors: Hupert-Kocurek Katarzyna , Sage Jay M. , Makowska-Grzyska Magdalena , Kaguni Jon M. 
Published: September 2007

ABSTRACT

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the
dnaC
gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of
Escherichia coli
and related enterobacteria. The method consists of using a strain carrying a large deletion of the
dnaC
gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-
d
-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a
dnaC
mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.
The Fungus-Specific HET Domain Mediates Programmed Cell Death in
Podospora anserina
Authors: Paoletti M. , Clavé C. 
Published: September 2007

ABSTRACT

Vegetative incompatibility is a programmed cell death reaction that occurs when fungal cells of unlike genotypes fuse. Genes defining vegetative incompatibility (
het
genes) are highly polymorphic, and most if not all incompatibility systems include a protein partner bearing the fungus-specific domain termed the HET domain. The nonallelic
het-C
/
het-E
incompatibility system is the best-characterized incompatibility system in
Podospora anserina
. Cell death is triggered by interaction of specific alleles of
het-C
, encoding a glycolipid transfer protein, and
het-E
, encoding a HET domain and a WD repeat domain involved in recognition. We show here that overexpression of the isolated HET domain from
het-E
results in cell death. This cell death is characterized by induction of autophagy, increased vacuolization, septation, and production of lipid droplets, which are hallmarks of cell death by incompatibility. In addition, the HET domain lethality is suppressed by the same mutations as vegetative incompatibility, but not by the inactivation of
het-C
. These results establish the HET domain as the mediator of cell death by incompatibility and lead to a modular conception of incompatibility systems whereby recognition is ensured by the variable regions of incompatibility proteins and cell death is triggered by the HET domain.
Expression of Hygromycin Phosphotransferase Alters Virulence of
Histoplasma capsulatum
Authors: Smulian A. George , Gibbons Reta S. , Demland Jeffery A. , Spaulding Deborah T. , Deepe George S. 
Published: September 2007

ABSTRACT

The
Escherichia coli
hygromycin phosphotransferase (
hph
) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the
hph
gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of
Histoplasma capsulatum
in the generation of knockout strains of
H. capsulatum
or as a marker in mutant strains.
hph
-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the
hph
gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of
H. capsulatum
. Infection of mice with
hph
-expressing
H. capsulatum
yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the
hph
construct into the genomes or to the method of integration and was not
H. capsulatum
strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the
hph
gene.
Supramolecular Organization of the Respiratory Chain in
Neurospora crassa
Mitochondria
Authors: Marques Isabel , Dencher Norbert A. , Videira Arnaldo , Krause Frank 
Published: September 2007

ABSTRACT

The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of
Neurospora crassa
, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of
N. crassa
comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three
Neurospora
mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.
Novel Membrane-Bound eIF2α Kinase in the Flagellar Pocket of
Trypanosoma brucei
Authors: Moraes Maria Carolina S. , Jesus Teresa C. L. , Hashimoto Nilce N. , Dey Madhusudan , Schwartz Kevin J. , Alves Viviane S. , Avila Carla C. , Bangs James D. , Dever Thomas E. , Schenkman Sergio , Castilho Beatriz A. 
Published: September 2007

ABSTRACT

Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations.
Trypanosoma brucei
, the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes.
T. brucei
eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in
T. brucei
, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes.
A Chemogenomic Screening of Sulfanilamide-Hypersensitive
Saccharomyces cerevisiae
Mutants Uncovers
ABZ2
, the Gene Encoding a Fungal Aminodeoxychorismate Lyase
Authors: Botet Javier , Mateos Laura , Revuelta José L. , Santos María A. 
Published: September 2007

ABSTRACT

Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the
p
-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (
YHI9
and
YMR289w
) were also identified. A detailed characterization of
YMR289w
revealed that this gene was required for growth in media lacking
p
-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for
p
-aminobenzoic acid biosynthesis. In light of these results,
YMR289w
was designated
ABZ2
, in accordance with the accepted nomenclature.
ABZ2
was able to rescue the
p
-aminobenzoate auxotrophy of an
Escherichia coli pabC
mutant, thus demonstrating that
ABZ2
and
pabC
are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.
Small Trypanosome RNA-Binding Proteins
Tb
UBP1 and
Tb
UBP2 Influence Expression of F-Box Protein mRNAs in Bloodstream Trypanosomes
Authors: Hartmann Claudia , Benz Corinna , Brems Stefanie , Ellis Louise , Luu Van-Duc , Stewart Mhairi , D'Orso Iván , Busold Christian , Fellenberg Kurt , Frasch Alberto C. C. , Carrington Mark , Hoheisel Jörg , Clayton Christine E. 
Published: September 2007

ABSTRACT

In the African trypanosome
Trypanosoma brucei
nearly all control of gene expression is posttranscriptional; sequences in the 3′-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins,
Tb
UBP1 and
Tb
UBP2, containing a single RNA recognition motif, in trypanosome gene expression.
Tb
UBP1 and
Tb
UBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of
Tb
UBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the
CFB1
mRNA, which encodes a protein with a cyclin F-box domain.
CFB1
regulation was mediated by the 3′-untranslated region and involved stabilization of the mRNA. Depletion of
Tb
UBP2 and
Tb
UBP1 inhibited growth and downregulated expression of the cyclin F box protein gene
CFB2
;
trans
splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to
Tb
UBP2 or
Tb
UBP1, with some preference for
CFB1
. We suggest that
Tb
UBP1 and
Tb
UBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.
Requirement for
Candida albicans
Sun41 in Biofilm Formation and Virulence
Authors: Norice Carmelle T. , Smith Frank J. , Solis Norma , Filler Scott G. , Mitchell Aaron P. 
Published: September 2007

ABSTRACT

The cell wall of
Candida albicans
lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in
SUN41
, Orf19.5412, Orf19.1277,
MSB2
, Orf19.3869, and
WSC1
caused hypersensitivity to the cell wall inhibitor caspofungin, while two different
ecm33
insertions caused mild caspofungin resistance. Insertion mutations in
SUN41
and Orf19.5412 caused biofilm defects. Through analysis of homozygous
sun41
Δ/
sun41
Δ deletion mutants and
sun41
Δ/
sun41
Δ+p
SUN41
-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The
sun41
Δ/
sun41
Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the
sun41
Δ/
sun41
Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.
Control of Cell Wall Assembly by a Histone-Like Protein in Mycobacteria
Authors: Katsube Tomoya , Matsumoto Sohkichi , Takatsuka Masaki , Okuyama Megumi , Ozeki Yuriko , Naito Mariko , Nishiuchi Yukiko , Fujiwara Nagatoshi , Yoshimura Mamiko , Tsuboi Takafumi , Torii Motomi , Oshitani Nobuhide , Arakawa Tetsuo , Kobayashi Kazuo 
Published: September 2007

ABSTRACT

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient
Mycobacterium smegmatis
strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.
Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted
Enterococcus faecium
Clonal Complex 17 Isolates
Authors: Hendrickx Antoni P. A. , van Wamel Willem J. B. , Posthuma George , Bonten Marc J. M. , Willems Rob J. L. 
Published: September 2007

ABSTRACT

Most
Enterococcus faecium
isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17
E. faecium
. Using PCR and Southern and dot blot hybridizations, 131
E. faecium
isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the
E. faecium
TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in
E. faecium
CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (
P
< 0.05). Three of these CWAP genes clustered together on the
E. faecium
TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering
E. faecium
contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.
Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe
Bacteroides fragilis
Authors: Rocha Edson R. , Tzianabos Arthur O. , Smith C. Jeffrey 
Published: September 2007

ABSTRACT

Results of this study showed that the anaerobic, opportunistic pathogen
Bacteroides fragilis
lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs. Analysis of the genome sequence revealed six Trx orthologs and an absence of genes required for synthesis of glutathione and glutaredoxins. In addition, it was shown that the thioredoxin reductase (TrxB)/Trx system is the major or sole redox system for thiol/disulfide cellular homeostasis in this anaerobic bacterium. Expression of the
B. fragilis trxB
gene was induced following treatment with diamide or H
2
O
2
or exposure to oxygen. This inducible
trxB
expression was OxyR independent. Northern blot hybridization analysis showed that the
trxB
mRNA was cotranscribed with
lolA
as a bicistronic transcript or was present as a monocistronic transcript that was also highly induced under the same conditions. The role of LolA, a prokaryotic periplasmic lipoprotein-specific molecular chaperone in the thiol/disulfide redox system, is unknown. A
trxB
deletion mutant was more sensitive to the effects of diamide and oxygen than the parent strain. In addition, the
trxB
mutant was unable to grow in culture media without addition of a reductant. Furthermore, the
trxB
mutant was not able to induce intraabdominal abscess formation in a mouse model, whereas the parent strain was. Taken together, these data strongly suggest that TrxB/Trx is the major, if not the sole, thiol/disulfide redox system in this anaerobe required for survival and abscess formation in a peritoneal cavity infection model.
Phosphopantetheine Adenylyltransferase from
Escherichia coli
: Investigation of the Kinetic Mechanism and Role in Regulation of Coenzyme A Biosynthesis
Authors: Miller J. Richard , Ohren Jeffrey , Sarver Ronald W. , Mueller W. Thomas , Dreu Piet de , Case Heather , Thanabal Venkataraman 
Published: September 2007

ABSTRACT

Phosphopantetheine adenylyltransferase (PPAT) from
Escherichia coli
is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in
E. coli
, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.
Structure and Biological Activities of Beta Toxin fromStaphylococcus aureus
Authors: Huseby Medora , Shi Ke , Brown C. Kent , Digre Jeff , Mengistu Fikre , Seo Keun Seok , Bohach Gregory A. , Schlievert Patrick M. , Ohlendorf Douglas H. , Earhart Cathleen A. 
Published: September 2007

ABSTRACTBeta toxin is a neutral sphingomyelinase secreted by certain strains ofStaphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases ofListeria ivanoviiandBacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1,Escherichia coliendonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 fromBombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.