bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomewide Location Analysis of
Candida albicans
Upc2p, a Regulator of Sterol Metabolism and Azole Drug Resistance
Authors: Znaidi Sadri , Weber Sandra , Zin Al-Abdin Osman , Bomme Perrine , Saidane Saloua , Drouin Simon , Lemieux Sébastien , De Deken Xavier , Robert François , Raymond Martine 
Published: April 2008

ABSTRACT

Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in
Candida albicans
. To better understand Upc2p function in
C. albicans
, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters (
P
< 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (
NCP1
,
ERG11
,
ERG2
, and others), 18 genes encoding ribosomal subunits (
RPS30
,
RPL32
,
RPL12
, and others), 3 genes encoding drug transporters (
CDR1
,
MDR1
, and
YOR1
), 4 genes encoding transcription factors (
INO2
,
ACE2
,
SUT1
, and
UPC2
), and 6 genes involved in sulfur amino acid metabolism (
MET6
,
SAM2
,
SAH1
, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5′-VNCGBDTR that includes the previously characterized Upc2p binding site 5′-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (
ERG11
,
MDR1
,
CDR1
,
YOR1
,
SUT1
,
SMF12
, and
CBP1
). The analysis of
ERG11
,
MDR1
, and
CDR1
transcripts in wild-type and
upc2
Δ/
upc2
Δ strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.
The Nuclear Migration Protein NUDF/LIS1 Forms a Complex with NUDC and BNFA at Spindle Pole Bodies
Authors: Helmstaedt Kerstin , Laubinger Karen , Voßkuhl Katja , Bayram Özgür , Busch Silke , Hoppert Michael , Valerius Oliver , Seiler Stephan , Braus Gerhard H. 
Published: April 2008

ABSTRACTNuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC inAspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (forbinding toNUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.
Molecular Signals in the Trafficking of
Toxoplasma gondii
Protein MIC3 to the Micronemes
Authors: El Hajj Hiba , Papoin Julien , Cérède Odile , Garcia-Réguet Nathalie , Soête Martine , Dubremetz Jean-François , Lebrun Maryse 
Published: April 2008

ABSTRACT

The protozoan parasite
Toxoplasma gondii
is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of
T. gondii
infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.
Photoregulation of Cellular Morphology during Complementary Chromatic Adaptation Requires Sensor-Kinase-Class Protein RcaE in
Fremyella diplosiphon
Authors: Bordowitz Juliana R. , Montgomery Beronda L. 
Published: April 2008

ABSTRACT

We used wild-type UTEX481; SF33, a shortened-filament mutant strain that shows normal complementary chromatic adaptation pigmentation responses; and FdBk14, an RcaE-deficient strain that lacks light-dependent pigmentation responses, to investigate the molecular basis of the photoregulation of cellular morphology in the cyanobacterium
Fremyella diplosiphon
. Detailed microscopic and biochemical analyses indicate that RcaE is required for the photoregulation of cell and filament morphologies of
F. diplosiphon
in response to red and green light.
Mobilization of the
incQ
Plasmid R300B with a Chromosomal Conjugation System in
Salmonella enterica
Serovar Typhi
Authors: Baker Stephen , Pickard Derek , Whitehead Sally , Farrar Jeremy , Dougan Gordon 
Published: April 2008

ABSTRACT

Salmonella
pathogenicity island 7 (SPI-7) in
Salmonella enterica
serovar Typhi appears to be related to other genomic islands. Evidence suggests that SPI-7 is susceptible to spontaneous circularization, loss, and transposition. Here, we demonstrate that a region within SPI-7 has the ability to mobilize the small
incQ
plasmid R300B.
Norepinephrine Mediates Acquisition of Transferrin-Iron in
Bordetella bronchiseptica
Authors: Anderson Mark T. , Armstrong Sandra K. 
Published: April 2008

ABSTRACT

Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of
Bordetella bronchiseptica
in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the
Bordetella bfeA
enterobactin catechol xenosiderophore receptor gene, neither a
bfeA
mutant nor a
bfeR
regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a
tonB
mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The
B. bronchiseptica
genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of
55
Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates
B. bronchiseptica
iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.
The
Xenorhabdus nematophila nilABC
Genes Confer the Ability of
Xenorhabdus
spp. To Colonize
Steinernema carpocapsae
Nematodes
Authors: Cowles Charles E. , Goodrich-Blair Heidi 
Published: April 2008

ABSTRACT

Members of the
Steinernema
genus of nematodes are colonized mutualistically by members of the
Xenorhabdus
genus of bacteria. In nature,
Steinernema carpocapsae
nematodes are always found in association with
Xenorhabdus nematophila
bacteria. Thus, this interaction, like many microbe-host associations, appears to be species specific.
X. nematophila
requires the
nilA
,
nilB
, and
nilC
genes to colonize
S. carpocapsae
. In this work, we showed that of all the
Xenorhabdus
species examined, only
X. nematophila
has the
nilA
,
nilB
, and
nilC
genes. By exposing
S. carpocapsae
to other
Xenorhabdus
spp., we established that only
X. nematophila
is able to colonize
S. carpocapsae
; therefore, the
S. carpocapsae-X. nematophila
interaction is species specific. Further, we showed that introduction of the
nilA
,
nilB
, and
nilC
genes into other
Xenorhabdus
species enables them to colonize the same
S. carpocapsae
host tissue that is normally colonized by
X. nematophila
. Finally, sequence analysis supported the idea that the
nil
genes were horizontally acquired. Our findings indicate that a single genetic locus determines host specificity in this bacteria-animal mutualism and that host range expansion can occur through the acquisition of a small genetic element.
Pseudomonas aeruginosa
Twitching Motility-Mediated Chemotaxis towards Phospholipids and Fatty Acids: Specificity and Metabolic Requirements
Authors: Miller Rhea M. , Tomaras Andrew P. , Barker Adam P. , Voelker Dennis R. , Chan Edward D. , Vasil Adriana I. , Vasil Michael L. 
Published: April 2008

ABSTRACT

Pseudomonas aeruginosa
demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of
P. aeruginosa
(i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via β-oxidation and the glyoxylate shunt. It was discovered that
P. aeruginosa
exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase (Δ
aceA
) and malate synthase (Δ
aceB
), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of
P. aeruginosa
up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the Δ
aceA
and Δ
aceB
mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.
Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane
Authors: Kaserer Wallace A. , Jiang Xiaoxu , Xiao Qiaobin , Scott Daniel C. , Bauler Matthew , Copeland Daniel , Newton Salete M. C. , Klebba Phillip E. 
Published: April 2008

ABSTRACTWe created hybrid proteins to study the functions of TonB. We first fused the portion ofEscherichia coli tonBthat encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream fromE. coli malE(MalE-TonB69C). Production of MalE-TonB69C intonB+bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus toE. coliYcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.
Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in
Mycobacterium smegmatis
Authors: Dass Bob Kennedy M. , Sharma Ritu , Shenoy Avinash R. , Mattoo Rohini , Visweswariah Sandhya S. 
Published: April 2008

ABSTRACT

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3′,5′-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in
Mycobacterium smegmatis
during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in
Mycobacterium tuberculosis
(Rv1647) and
Mycobacterium leprae
(ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ΔMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type
M. smegmatis
under conditions of acid stress but not in the ΔMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ΔMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.
Characterization of a Novel Methanol Dehydrogenase in Representatives of
Burkholderiales
: Implications for Environmental Detection of Methylotrophy and Evidence for Convergent Evolution
Authors: Kalyuzhnaya Marina G. , Hristova Krassimira R. , Lidstrom Mary E. , Chistoserdova Ludmila 
Published: April 2008

ABSTRACT

Some members of
Burkholderiales
are able to grow on methanol but lack the genes (
mxaFI
) responsible for the well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydrogenase that is widespread in methylotrophic
Proteobacteria
. Here, we characterized novel, mono-subunit enzymes responsible for methanol oxidation in four strains,
Methyloversatilis universalis
FAM5,
Methylibium petroleiphilum
PM1, and unclassified
Burkholderiales
strains RZ18-153 and FAM1. The enzyme from
M. universalis
FAM5 was partially purified and subjected to matrix-assisted laser desorption ionization-time of fight peptide mass fingerprinting. The resulting peptide spectrum was used to identify a gene candidate in the genome of
M. petroleiphilum
PM1 (
mdh2
) predicted to encode a type I alcohol dehydrogenase related to the characterized methanol dehydrogenase large subunits but at less than 35% amino acid identity. Homologs of
mdh2
were amplified from
M. universalis
FAM5 and strains RZ18-153 and FAM1, and mutants lacking
mdh2
were generated in three of the organisms. These mutants lost their ability to grow on methanol and ethanol, demonstrating that
mdh2
is responsible for oxidation of both substrates. Our findings have implications for environmental detection of methylotrophy and indicate that this ability is widespread beyond populations possessing
mxaF
, the gene traditionally used as a genetic marker for environmental detection of methanol-oxidizing capability. Our findings also have implications for understanding the evolution of methanol oxidation, suggesting a convergence toward the enzymatic function for methanol oxidation in MxaF and Mdh2-type proteins.
Differential Transcriptional Analysis of the Cyanobacterium
Cyanothece
sp. Strain ATCC 51142 during Light-Dark and Continuous-Light Growth
Authors: Toepel Jörg , Welsh Eric , Summerfield Tina C. , Pakrasi Himadri B. , Sherman Louis A. 
Published: April 2008

ABSTRACT

We analyzed the metabolic rhythms and differential gene expression in the unicellular, diazotrophic cyanobacterium
Cyanothece
sp. strain ATCC 51142 under N
2
-fixing conditions after a shift from normal 12-h light-12-h dark cycles to continuous light. We found that the mRNA levels of ∼10% of the genes in the genome demonstrated circadian behavior during growth in free-running (continuous light) conditions. The genes for N
2
fixation displayed a strong circadian behavior, whereas photosynthesis and respiration genes were not as tightly regulated. One of our main objectives was to determine the strategies used by these cells to perform N
2
fixation under normal day-night conditions, as well as under the greater stress caused by continuous light. We determined that N
2
fixation cycled in continuous light but with a lower N
2
fixation activity. Glycogen degradation, respiration, and photosynthesis were also lower; nonetheless, O
2
evolution was about 50% of the normal peak. We also demonstrated that
nifH
(encoding the nitrogenase Fe protein),
nifB
, and
nifX
were strongly induced in continuous light; this is consistent with the role of these proteins during the assembly of the enzyme complex and suggested that the decreased N
2
fixation activity was due to protein-level regulation or inhibition. Many soluble electron carriers (e.g., ferredoxins), as well as redox carriers (e.g., thioredoxin and glutathione), were strongly induced during N
2
fixation in continuous light. We suggest that these carriers are required to enhance cyclic electron transport and phosphorylation for energy production and to maintain appropriate redox levels in the presence of elevated O
2
, respectively.
Growth ofEscherichia coli: Significance of Peptidoglycan Degradation during Elongation and Septation
Authors: Uehara Tsuyoshi , Park James T. 
Published: April 2008

ABSTRACTWe have found a striking difference between the modes of action of amdinocillin (mecillinam) and compound A22, both of which inhibit cell elongation. This was made possible by employment of a new method using anEscherichia colipeptidoglycan (PG)-recycling mutant, lackingampD, to analyze PG degradation during cell elongation and septation. Using this method, we have found that A22, which is known to prevent MreB function, strongly inhibited PG synthesis during elongation. In contrast, treatment of elongating cells with amdinocillin, which inhibits penicillin-binding protein 2 (PBP2), allowed PG glycan synthesis to proceed at a nearly normal rate with concomitant rapid degradation of the new glycan strands. By treating cells with A22 to inhibit sidewall synthesis, the method could also be applied to study septum synthesis. To our surprise, over 30% of newly synthesized septal PG was degraded during septation. Thus, excess PG sufficient to form at least one additional pole was being synthesized and rapidly degraded during septation. We propose that during cell division, rapid removal of the excess PG serves to separate the new poles of the daughter cells. We have also employed this new method to demonstrate that PBP2 and RodA are required for the synthesis of glycan strands during elongation and that the periplasmic amidases that aid in cell separation are minor players, cleaving only one-sixth of the PG that is turned over by the lytic transglycosylases.
Insertion Hot Spot for Horizontally Acquired DNA within a Bidirectional Small-RNA Locus inSalmonella enterica
Authors: Balbontín Roberto , Figueroa-Bossi Nara , Casadesús Josep , Bossi Lionello 
Published: April 2008

ABSTRACTInEscherichia coliandSalmonella enterica, RyeA and RyeB RNAs are encoded on opposite DNA strands at the same locus. We present evidence indicating that the last 23 bp of theryeBgene, corresponding to an internal portion of theryeAgene, served repeatedly as the integration site for exogenous DNA duringSalmonellaevolution and still act as an attachment site for present-day bacteriophages. Interestingly,ryeAsequence and expression are modified upon lysogenization.
The
Methanothermobacter thermautotrophicus
Cdc6-2 Protein, the Putative Helicase Loader, Dissociates the Minichromosome Maintenance Helicase
Authors: Shin Jae-Ho , Heo Gun Young , Kelman Zvi 
Published: April 2008

ABSTRACT

The Cdc6-1 and -2 proteins from the archaeon
Methanothermobacter thermautotrophicus
were previously shown to bind the minichromosome maintenance (MCM) helicase. It is shown here that Cdc6-2 protein dissociates the MCM complex. This observation supports the hypothesis that the Cdc6-2 protein functions as a helicase loader.