bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach
Authors: Blazeck John , Liu Leqian , Redden Heidi , Alper Hal 
Published: September 2011

ABSTRACT

The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast
Yarrowia lipolytica
. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported for
Y. lipolytica
. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters in
Y. lipolytica
are enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.
Beta Hydroxylation of Glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-Dependent β-Hydroxylase
Authors: Teichmann Beate , Lefebvre François , Labbé Caroline , Bölker Michael , Linne Uwe , Bélanger Richard R. 
Published: September 2011

ABSTRACT

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by
Pseudozyma flocculosa
, a biocontrol agent, and
Ustilago maydis
, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the
P. flocculosa fhd1
gene and its homolog from
U. maydis
,
uhd1
, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the
uhd1
gene in
U. maydis
and analyzed the secreted UA.
uhd1
deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of
P. flocculosa
Fhd1, the corresponding gene was used to complement
U. maydis
Δ
uhd1
mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a
P. flocculosa
homolog of the
U. maydis
long-chain fatty-acid alpha hydroxylase Ahd1. The
P. flocculosa ahd1
gene, which does not reside in the flocculosin gene cluster, was introduced into
U. maydis
Δ
ahd1
mutant strains.
P. flocculosa
Ahd1 neither complemented the
U. maydis
Δ
ahd1
phenotype nor resulted in the production of β-hydroxylated UA. This suggests that
P. flocculosa
Ahd1 is not involved in flocculosin hydroxylation.
Structural and Spatial Associations between Fe, O, and C in the Network Structure of the Leptothrix ochracea Sheath Surface
Authors: Suzuki Tomoko , Hashimoto Hideki , Ishihara Hiromichi , Kasai Tomonari , Kunoh Hitoshi , Takada Jun 
Published: September 2011

ABSTRACT

The structural and spatial associations of Fe with O and C in the outer coat fibers of the
Leptothrix ochracea
sheath were shown to be substantially similar to the stalk fibers of
Gallionella ferruginea
, i.e., a central C core, probably of bacterial origin, and aquatic Fe interacting with O at the surface of the core.
Examination of the Seasonal Dynamics of the Toxic Dinoflagellate Alexandrium catenella at Redondo Beach, California, by Quantitative PCR
Authors: Garneau Marie-Ève , Schnetzer Astrid , Countway Peter D. , Jones Adriane C. , Seubert Erica L. , Caron David A. 
Published: September 2011

ABSTRACT

The presence of neurotoxic species within the genus
Alexandrium
along the U.S. coastline has raised concern of potential poisoning through the consumption of contaminated seafood. Paralytic shellfish toxins (PSTs) detected in shellfish provide evidence that these harmful events have increased in frequency and severity along the California coast during the past 25 years, but the timing and location of these occurrences have been highly variable. We conducted a 4-year survey in King Harbor, CA, to investigate the seasonal dynamics of
Alexandrium catenella
and the presence of a particulate saxitoxin (STX), the parent compound of the PSTs. A quantitative PCR (qPCR) assay was developed for quantifying
A. catenella
in environmental microbial assemblages. This approach allowed for the detection of abundances as low as 12 cells liter
−1
, 2 orders of magnitude below threshold abundances that can impact food webs.
A. catenella
was found repeatedly during the study, particularly in spring, when cells were detected in 38% of the samples (27 to 5,680 cells liter
−1
). This peak in cell abundances was observed in 2006 and corresponded to a particulate STX concentration of 12 ng liter
−1
, whereas the maximum STX concentration of 26 ng liter
−1
occurred in April 2008. Total cell abundances and toxin levels varied strongly throughout each year, but
A. catenella
was less abundant during summer, fall, and winter, when only 2 to 11% of the samples yielded positive qPCR results. The qPCR method developed here provides a useful tool for investigating the ecology of
A. catenella
at subbloom and bloom abundances.
Stable Isotope Probing of an Algal Bloom To Identify Uncultivated Members of the Rhodobacteraceae Associated with Low-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation
Authors: Gutierrez Tony , Singleton David R. , Aitken Michael D. , Semple Kirk T. 
Published: September 2011

ABSTRACT

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria associated with an algal bloom in Tampa Bay, FL, were investigated by stable isotope probing (SIP) with uniformly labeled [
13
C]naphthalene. The dominant sequences in clone libraries constructed from
13
C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the family
Rhodobacteraceae
. Quantitative PCR primers targeting the 16S rRNA gene of these uncultivated organisms were used to determine their abundance in incubations amended with unlabeled naphthalene and phenanthrene, both of which showed substantial increases in gene copy numbers during the experiments. As demonstrated by this work, the application of uniformly
13
C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters.
Bacterium-Induced Internal Egg Hatching Frequency Is Predictive of Life Span in Caenorhabditis elegans Populations
Authors: Mosser Thomas , Matic Ivan , Leroy Magali 
Published: September 2011

ABSTRACT

Internal egg hatching in
Caenorhabditis elegans
, “worm bagging,” is induced by exposure to bacteria. This study demonstrates that the determination of worm bagging frequency allows for advanced insight into the degree of bacterial pathogenicity and is highly predictive of the survival of worm populations. Therefore, worm bagging frequency can be regarded as a reliable population-wide stress reporter.
Putative Stress Sensors WscA and WscB Are Involved in Hypo-Osmotic and Acidic pH Stress Tolerance in Aspergillus nidulans
Authors: Futagami Taiki , Nakao Seiki , Kido Yayoi , Oka Takuji , Kajiwara Yasuhiro , Takashita Hideharu , Omori Toshiro , Furukawa Kensuke , Goto Masatoshi 
Published: September 2011

ABSTRACT

Wsc proteins have been identified in fungi and are believed to be stress sensors in the cell wall integrity (CWI) signaling pathway. In this study, we characterized the sensor orthologs WscA and WscB in
Aspergillus nidulans
. Using hemagglutinin-tagged WscA and WscB, we showed both Wsc proteins to be
N
- and
O
-glycosylated and localized in the cell wall and membrane, implying that they are potential cell surface sensors. The
wscA
disruptant (Δ
wscA
) strain was characterized by reduced colony and conidia formation and a high frequency of swollen hyphae under hypo-osmotic conditions. The deficient phenotype of the Δ
wscA
strain was facilitated by acidification, but not by alkalization or antifungal agents. In contrast, osmotic stabilization restored the normal phenotype in the Δ
wscA
strain. A similar inhibition was observed in the
wscB
disruptant strain, but to a lesser extent. In addition, a double
wscA
and
wscB
disruptant (Δ
wscA
Δ
wscB
) strain was viable, but its growth was inhibited to a greater degree, indicating that the functions of the products of these genes are redundant. Transcription of
α
-1,3-glucan synthase genes (
agsA
and
agsB
) was significantly altered in the
wscA
disruptant strain, resulting in an increase in the amount of alkali-soluble cell wall glucan compared to that in the wild-type (wt) strain. An increase in mitogen-activated protein kinase (MpkA) phosphorylation was observed as a result of
wsc
disruption. Moreover, the transient transcriptional upregulation of the
agsB
gene via MpkA signaling was observed in the Δ
wscA
Δ
wscB
strain to the same degree as in the wt strain. These results indicate that
A. nidulans
Wsc proteins have a different sensing spectrum and downstream signaling pathway than those in the yeast
Saccharomyces cerevisiae
and that they play an important role in CWI under hypo-osmotic and acidic pH conditions.
Morphological Events during the Cell Cycle of Leishmania major
Authors: Ambit Audrey , Woods Kerry L. , Cull Benjamin , Coombs Graham H. , Mottram Jeremy C. 
Published: September 2011

ABSTRACT

The morphological events involved in the
Leishmania major
promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling of β-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used to determine the durations of phases of the cell cycle. Our results demonstrate that in
L. major
promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute to a more complete foundation for future studies of cell cycle control in
Leishmania
.
Mitochondria and Fungal Pathogenesis: Drug Tolerance, Virulence, and Potential for Antifungal Therapy
Authors: Shingu-Vazquez Miguel , Traven Ana 
Published: September 2011

ABSTRACT
Recently, mitochondria have been identified as important contributors to the virulence and drug tolerance of human fungal pathogens. In different scenarios, either hypo- or hypervirulence can result from changes in mitochondrial function. Similarly, specific mitochondrial mutations lead to either sensitivity or resistance to antifungal drugs. Here, we provide a synthesis of this emerging field, proposing that mitochondrial function in membrane lipid homeostasis is the common denominator underlying the observed effects of mitochondria in drug tolerance (both sensitivity and resistance). We discuss how the contrasting effects of mitochondrial dysfunction on fungal drug tolerance and virulence could be explained and the potential for targeting mitochondrial factors for future antifungal drug development.
Epigenetic Regulation of Polymerase II Transcription Initiation in Trypanosoma cruzi: Modulation of Nucleosome Abundance, Histone Modification, and Polymerase Occupancy by O-Linked Thymine DNA Glucosylation
Authors: Ekanayake Dilrukshi , Sabatini Robert 
Published: September 2011

ABSTRACT

Very little is understood regarding how transcription is initiated/regulated in the early-diverging eukaryote
Trypanosoma cruzi
. Unusually for a eukaryote, genes transcribed by RNA polymerase (Pol) II in
T. cruzi
are arranged in polycistronic transcription units (PTUs). On the basis of this gene organization, it was previously thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. We recently localized a novel glucosylated thymine DNA base, called base J, to potential promoter regions of PTUs throughout the trypanosome genome. Loss of base J, following the deletion of JBP1, a thymidine hydroxylase involved with synthesis, led to a global increase in the Pol II transcription rate and gene expression. In order to determine the mechanism by which base J regulates transcription, we have characterized changes in chromatin structure and Pol II recruitment to promoter regions following the loss of base J. The loss of base J coincides with a decrease in nucleosome abundance, increased histone H3/H4 acetylation, and increased Pol II occupancy at promoter regions, including the well-characterized spliced leader RNA gene promoter. These studies present the first direct evidence for epigenetic regulation of Pol II transcription initiation via DNA modification and chromatin structure in kinetoplastids as well as provide a mechanism for regulation of trypanosome gene expression via the novel hypermodified base J.
Branch Point Identification and Sequence Requirements for Intron Splicing in Plasmodium falciparum
Authors: Zhang Xiaohong , Tolzmann Caitlin A. , Melcher Martin , Haas Brian J. , Gardner Malcolm J. , Smith Joseph D. , Feagin Jean E. 
Published: September 2011

ABSTRACT

Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary.
Plasmodium falciparum
has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an
in vivo
luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5′ and 3′ splicing requirements and experimentally determine the intron branch point in
P. falciparum
. This analysis indicates that
P. falciparum
mRNAs have canonical 5′ and 3′ splice sites. However, the 5′ consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3′ splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different
P. falciparum
pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that
P. falciparum
retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack.
The Casein Kinase I Protein Cck1 Regulates Multiple Signaling Pathways and Is Essential for Cell Integrity and Fungal Virulence in Cryptococcus neoformans
Authors: Wang Yina , Liu Tong-Bao , Patel Shyam , Jiang Linghuo , Xue Chaoyang 
Published: September 2011

ABSTRACT

Casein kinases regulate a wide range of cellular functions in eukaryotes, including phosphorylation of proteins that are substrates for degradation via the ubiquitin-proteasome system (UPS). Our previous study demonstrated that Fbp1, a component of the SCF
FBP1
E3 ligase complex, was essential for
Cryptococcus
virulence. Because the
Saccharomyces cerevisiae
homolog of Fbp1, Grr1, requires casein kinase I (Yck1 and Yck2) to phosphorylate its substrates, we investigated the function of casein kinase I in
Cryptococcus neoformans
. In this report, we identified a
C. neoformans
casein kinase I protein homolog, Cck1. Similar to Fbp1, the expression of Cck1 is negatively regulated by glucose and during mating.
cck1
null mutants showed significant virulence attenuation in a murine systemic infection model, but Cck1 was dispensable for the development of classical virulence factors (capsule, melanin, and growth at 37°C).
cck1
mutants were hypersensitive to SDS treatment, indicating that Cck1 is required for cell integrity. The functional overlap between Cck1 and Fbp1 suggests that Cck1 may be required for the phosphorylation of Fbp1 substrates. Interestingly, the
cck1
mutant also showed increased sensitivity to osmotic stress and oxidative stress, suggesting that Cck1 regulates both cell integrity and the cellular stress response. Our results show that Cck1 regulates the phosphorylation of both Mpk1 and Hog1 mitogen-activated protein kinases (MAPKs), demonstrating that Cck1 regulates cell integrity via the Mpk1 pathway and regulates cell adaptation to stresses via the Hog1 pathway. Overall, our study revealed that Cck1 plays important roles in regulating multiple signaling pathways and is required for fungal pathogenicity.
Integration of Cyclic di-GMP and Quorum Sensing in the Control of
vpsT
and
aphA
in Vibrio cholerae
Authors: Srivastava Disha , Harris Rebecca C. , Waters Christopher M. 
Published: September 2011

ABSTRACT

Vibrio cholerae
transitions between aquatic environmental reservoirs and infection in the gastrointestinal tracts of human hosts. The second-messenger molecule cyclic di-GMP (c-di-GMP) and quorum sensing (QS) are important signaling systems that enable
V. cholerae
to alternate between these distinct environments by controlling biofilm formation and virulence factor expression. Here we identify a conserved regulatory mechanism in
V. cholerae
that integrates c-di-GMP and QS to control the expression of two transcriptional regulators:
aphA
, an activator of virulence gene expression and an important regulator of the quorum-sensing pathway, and
vpsT
, a transcriptional activator that induces biofilm formation. Surprisingly,
aphA
expression was induced by c-di-GMP. Activation of both
aphA
and
vpsT
by c-di-GMP requires the transcriptional activator VpsR, which binds to c-di-GMP. The VpsR binding site at each of these promoters overlaps with the binding site of HapR, the master QS regulator at high cell densities. Our results suggest that
V. cholerae
combines information conveyed by QS and c-di-GMP to appropriately respond and adapt to divergent environments by modulating the expression of key transcriptional regulators.
FadD Is Required for Utilization of Endogenous Fatty Acids Released from Membrane Lipids
Authors: Pech-Canul Ángel , Nogales Joaquina , Miranda-Molina Alfonso , Álvarez Laura , Geiger Otto , Soto María José , López-Lara Isabel M. 
Published: September 2011

ABSTRACT

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of
fadD
in the symbiotic nitrogen-fixing bacterium
Sinorhizobium meliloti
promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that
S. meliloti
fadD
mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids.
Escherichia coli
fadD
mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of
E. coli
with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in
fadD
mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with
S. meliloti
revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.
Deciphering Morphological Determinants of the Helix-Shaped Leptospira
Authors: Slamti Leyla , de Pedro Miguel A. , Guichet Emilande , Picardeau Mathieu 
Published: September 2011

ABSTRACT

Leptospira
spp. are thin, highly motile, slow-growing spirochetes that can be distinguished from other bacteria on the basis of their unique helical shape. Defining the mechanisms by which these bacteria generate and maintain this atypical morphology should greatly enhance our understanding of the fundamental physiology of these pathogens. In this study, we showed that peptidoglycan sacculi from
Leptospira
spp. retain the helical shape of intact cells. Interestingly, the distribution of muropeptides was different from that in the
Escherichia coli
model, indicating that specific enzymes might be active on the peptidoglycan macromolecule. We could alter the shape of
Leptospira biflexa
with the broad-spectrum β-lactam antibiotic penicillin G and with amdinocillin and aztreonam, which are β-lactams that preferentially target penicillin-binding protein 2 (PBP2) and PBP3, respectively, in some species. Although genetic manipulations of
Leptospira
spp. are scarce, we were able to obtain mutants with alterations in genes encoding PBPs, including PBP3. Loss of this protein resulted in cell elongation. We also generated an
L. biflexa
strain that conditionally expresses MreB. Loss of the MreB function was correlated with morphological abnormalities such as a localized increased diameter and heterogeneous length. A prolonged depletion of MreB resulted in cell lysis, suggesting that this protein is essential. These findings indicate that important aspects of leptospiral cell morphology are determined by the cytoskeleton and the murein layer, thus providing a starting point for a better understanding of the morphogenesis in these atypical bacteria.