bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Virulence Factors by Staphylococcus aureus Grown in Serum
Authors: Oogai Yuichi , Matsuo Miki , Hashimoto Masahito , Kato Fuminori , Sugai Motoyuki , Komatsuzawa Hitoshi
Published: September 2011

Staphylococcus aureus
produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by
in vitro
experiments using bacterial medium. However, when
S. aureus
infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in
S. aureus
grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl
3
into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with
agr
inactivated grown in serum, the expression of RNA III,
psm
, and
sec4
was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that
S. aureus
expresses virulence factors in adaptation to the host environment.
Staphylococcus epidermidis Bacteriophages from the Anterior Nares of Humans
Authors: Aswani Vijay , Tremblay Denise M. , Moineau Sylvain , Shukla Sanjay K.
Published: September 2011

The role of virulent bacteriophages in staphylococcal colonization of the human anterior nares is not known. This report of lytic bacteriophages against
Staphylococcus epidermidis
in the anterior nares of 5.5% of human subjects (
n
= 202) suggests their potential role in modulating staphylococcal colonization in this ecological niche.
Fimbrial Adhesins Produced by Atypical Enteropathogenic Escherichia coli Strains
Authors: Hernandes Rodrigo T. , Velsko Irina , Sampaio Suely C. F. , Elias Waldir P. , Robins-Browne Roy M. , Gomes Tï¿½nia A. T. , Girï¿½n Jorge A.
Published: September 2011

Atypical enteropathogenic
Escherichia coli
(aEPEC) has emerged as a significant cause of pediatric diarrhea worldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. In this study, we investigated the prevalence of several (
fimA
,
ecpA
,
csgA
,
elfA
, and
hcpA
) fimbrial genes in 71 aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Brazil and Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic and commensal
E. coli
. Here, the most prevalent fimbrial genes found, in descending order, were
hcpA
(98.6%),
ecpA
(86%),
fimA
(76%),
elfA
(72%), and
csgA
(19.7%). Phenotypic expression of pili in aEPEC strains was assessed by several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the
E. coli
laminin-binding fimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in 59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains, respectively. The
E. coli
common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering to HeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence for some aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strains involving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins and indicates that these strains bear several pilus operons that could potentially be expressed in different niches favoring colonization and survival in and outside the host.
Correlation between Protection against Sepsis by Probiotic Therapy and Stimulation of a Novel Bacterial Phylotype
Authors: Gerritsen Jacoline , Timmerman Harro M. , Fuentes Susana , van Minnen L. Paul , Panneman Henk , Konstantinov Sergey R. , Rombouts Frans M. , Gooszen Hein G. , Akkermans Louis M. A. , Smidt Hauke , Rijkers Ger T.
Published: September 2011

Prophylactic probiotic therapy has shown beneficial effects in an experimental rat model for acute pancreatitis on the health status of the animals. Mechanisms by which probiotic therapy interferes with severity of acute pancreatitis and associated sepsis, however, are poorly understood. The aims of this study were to identify the probiotic-induced changes in the gut microbiota and to correlate these changes to disease outcome. Duodenum and ileum samples were obtained from healthy and diseased rats subjected to pancreatitis for 7 days and prophylactically treated with either a multispecies probiotic mixture or a placebo. Intestinal microbiota was characterized by terminal-restriction fragment length polymorphism (T-RFLP) analyses of PCR-amplified 16S rRNA gene fragments. These analyses showed that during acute pancreatitis the host-specific ileal microbiota was replaced by an “acute pancreatitis-associated microbiota.” This replacement was not reversed by administration of the probiotic mixture. An increase, however, was observed in the relative abundance of a novel bacterial phylotype most closely related to
Clostridium lituseburense
and referred to as commensal rat ileum bacterium (CRIB). Specific primers targeting the CRIB 16S rRNA gene sequence were developed to detect this phylotype by quantitative PCR. An ileal abundance of CRIB 16S rRNA genes of more than 7.5% of the total bacterial 16S rRNA gene pool was correlated with reduced duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, improved pancreas pathology, and reduced proinflammatory cytokine levels in plasma. Our current findings and future studies involving this uncharacterized bacterial phylotype will contribute to unraveling one of the potential mechanisms of probiotic therapy.
Species Diversity, Community Dynamics, and Metabolite Kinetics of the Microbiota Associated with Traditional Ecuadorian Spontaneous Cocoa Bean Fermentations
Authors: Papalexandratou Zoi , Falony Gwen , Romanens Edwina , Jimenez Juan Carlos , Amores Freddy , Daniel Heide-Marie , De Vuyst Luc
Published: September 2011

Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species werePichia kudriavzeviiandPichia manshurica, followed bySaccharomyces cerevisiae. Leuconostoc pseudomesenteroides(glucose and fructose fermenting),Fructobacillus tropaeoli-like (fructose fermenting), andLactobacillus fermentum(citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates.Tatumella saanichensisandTatumella punctatawere among the members of the familyEnterobacteriaceaepresent during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely,Candida sorbosivorans-like. Acetic acid bacteria, whose main representative wasAcetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.
Development of
phoH
as a Novel Signature Gene for Assessing Marine Phage Diversity
Authors: Goldsmith Dawn B. , Crosti Giuseppe , Dwivedi Bhakti , McDaniel Lauren D. , Varsani Arvind , Suttle Curtis A. , Weinbauer Markus G. , Sandaa Ruth-Anne , Breitbart Mya
Published: September 2011

Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified,
phoH
was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage
phoH
sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the
phoH
gene were used to determine the diversity of phage
phoH
sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide.
phoH
was present at all sites examined, and a high diversity of
phoH
sequences was recovered. Most
phoH
sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct
phoH
composition, although most
phoH
clusters were recovered from multiple sites. Overall,
phoH
is an effective signature gene for examining phage diversity in the marine environment.
rnr
Gene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R
Authors: Sulthana Shaheen , Rajyaguru Purusharth I. , Mittal Pragya , Ray Malay K.
Published: September 2011

RNase R is a highly processive, hydrolytic 3′-5′ exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacterium
Pseudomonas syringae
Lz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (
rnr
) locus from this bacterium. By cloning and expressing a His
6
-tagged form of the
P. syringae
RNase R (RNase R
Ps
), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg
2+
and Mn
2+
) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase R
Ps
exhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structured
malE-malF
RNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5′-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain by
Escherichia coli
RNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase R
Ps
in vitro
. The ability of the nonhydrolyzable ATP-γS to inhibit RNase R
Ps
activity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.
Dynamics of Microbial Community Structure of and Enhanced Biological Phosphorus Removal by Aerobic Granules Cultivated on Propionate or Acetate
Authors: Gonzalez-Gil Graciela , Holliger Christof
Published: September 2011

Aerobic granules are dense microbial aggregates with the potential to replace floccular sludge for the treatment of wastewaters. In bubble-column sequencing batch reactors, distinct microbial populations dominated propionate- and acetate-cultivated aerobic granules after 50 days of reactor operation when only carbon removal was detected. Propionate granules were dominated by
Zoogloea
(40%),
Acidovorax
, and
Thiothrix
, whereas acetate granules were mainly dominated by
Thiothrix
(60%). Thereafter, an exponential increase in enhanced biological phosphorus removal (EBPR) activity was observed in the propionate granules, but a linear and erratic increase was detected in the acetate ones. Besides
Accumulibacter
and
Competibacter
, other bacterial populations found in both granules were associated with
Chloroflexus
and
Acidovorax
. The EBPR activity in the propionate granules was high and stable, whereas EBPR in the acetate granules was erratic throughout the study and suffered from a deterioration period that could be readily reversed by inducing hydrolysis of polyphosphate in presumably saturated
Accumulibacter
cells. Using a new
ppk1
gene-based dual terminal-restriction fragment length polymorphism (T-RFLP) approach revealed that
Accumulibacter
diversity was highest in the floccular sludge inoculum but that when granules were formed, propionate readily favored the dominance of
Accumulibacter
type IIA. In contrast, acetate granules exhibited transient shifts between type I and type II before the granules were dominated by
Accumulibacter
type IIA. However,
ppk1
gene sequences from acetate granules clustered separately from those of propionate granules. Our data indicate that the mere presence of
Accumulibacter
is not enough to have consistently high EBPR but that the type of
Accumulibacter
determines the robustness of the phosphate removal process.
Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach
Authors: Blazeck John , Liu Leqian , Redden Heidi , Alper Hal
Published: September 2011

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Cyclic AMP Receptor Protein Regulates
cspE
, an Early Cold-Inducible Gene, in Escherichia coli
Authors: Uppal Sheetal , Maurya Svetlana R. , Hire Ramesh S. , Jawali Narendra
Published: September 2011

cspE
, a member of the
cspA
family of cold shock proteins in
Escherichia coli
, is an early cold-inducible protein. The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulates
cspE
in
E. coli
. Sequence analysis of the
cspE
upstream region revealed a putative CRP target site centered at −61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for P

cspE

activation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-P

cspE

interaction. Based on the promoter architecture, we classified P

cspE

as a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in P

cspE

transcription. Furthermore, the substitutions in the key residues of the RNA polymerase α-subunit C-terminal domain (α-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in P

cspE

transcription. In addition, the deletion of
crp
led to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.
The CgrA and CgrC Proteins Form a Complex That Positively Regulates
cupA
Fimbrial Gene Expression in Pseudomonas aeruginosa
Authors: McManus Heather R. , Dove Simon L.
Published: September 2011

The CgrA and CgrC proteins of
Pseudomonas aeruginosa
are coregulators that are required for the phase-variable expression of the
cupA
fimbrial genes. Neither CgrA nor CgrC resembles a classical transcription regulator, and precisely how these proteins exert their regulatory effects on
cupA
gene expression is poorly understood. Here, we show that CgrA and CgrC interact with one another directly. We identify a mutant of CgrC that is specifically defective for interaction with CgrA and demonstrate that this mutant cannot restore the phase-variable expression of the
cupA
fimbrial genes to cells of a
cgrC
mutant strain. Using this mutant, we also show that CgrC associates with the
cupA
promoter regardless of whether or not it interacts with CgrA. Our findings establish that interaction between CgrA and CgrC is required for the phase-variable expression of the
cupA
fimbrial genes and suggest that CgrC exerts its regulatory effects directly at the
cupA
promoter, possibly by recruiting CgrA. Because the regions of CgrA and CgrC that we have identified as interacting with one another are highly conserved among orthologs, our findings raise the possibility that CgrA- and CgrC-related regulators present in other bacteria function coordinately through a direct protein-protein interaction.
RpiR Homologues May Link Staphylococcus aureus RNAIII Synthesis and Pentose Phosphate Pathway Regulation
Authors: Zhu Yefei , Nandakumar Renu , Sadykov Marat R. , Madayiputhiya Nandakumar , Luong Thanh T. , Gaupp Rosmarie , Lee Chia Y. , Somerville Greg A.
Published: September 2011

Staphylococcus aureus
is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the
rpiR
homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes
rpiA
and
zwf
, while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the
agr
quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription.
Bacillus subtilis σ
V
Confers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and
d
-Alanylation of Teichoic Acids
Authors: Guariglia-Oropeza Veronica , Helmann John D.
Published: September 2011

The seven extracytoplasmic function (ECF) sigma (σ) factors of
Bacillus subtilis
are broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σ
V
as including at least 20 operons, many of which are also regulated by σ
M
, σ
X
, or σ
W
. The σ
V
regulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance of
B. subtilis
to lysozyme. Strains with null mutations in either
sigV
or all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σ
V
in the Δ7ECF background restores lysozyme resistance, whereas induction of σ
M
, σ
X
, or σ
W
does not. Lysozyme resistance results from the ability of σ
V
to activate the transcription of two operons: the autoregulated
sigV
-
rsiV
-
oatA
-
yrhK
operon and
dltABCDE
. Genetic analyses reveal that
oatA
and
dlt
are largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas an
oatA dltA
double mutant is as sensitive as a
sigV
null strain. Moreover, the
sigV oatA dltA
triple mutant is no more sensitive than the
oatA dltA
double mutant, indicating that there are no other σ
V
-dependent genes necessary for lysozyme resistance. Thus, we suggest that σ
V
confers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA and
d
-alanylation of teichoic acids by DltABCDE.
Attempting to explain heterogeneous HIV epidemics in sub-Saharan Africa: potential role of historical changes in risk behaviour and male circumcision
Authors: Orroth Kate K , White Richard G , Freeman Esther E , Bakker Roel , Buvé Anne , Glynn Judith R , Habbema J Dik F , Hayes Richard J
Published: September 2011

Background

A key conclusion of the Four Cities Study, carried out to explore reasons for heterogeneity in the HIV epidemic between two cities in sub-Saharan Africa with relatively low prevalence (Cotonou and Yaoundé) and two with high prevalence (Kisumu and Ndola), was that differences in biological cofactors outweighed differences in sexual risk behaviours. The authors explore an alternative hypothesis, that risk behaviours were historically higher in the high-prevalence cities. They also investigate the effects of different prevalence of male circumcision on the HIV epidemics in the four cities.

Methods

A transmission model was fitted to data from the Four Cities Study. Default scenarios included biological cofactor effects on HIV transmission. Counter-factual scenarios were simulated without biological cofactors, with and without higher historical sexual behaviours, and with various rates of male circumcision.

Results

Simulated adult HIV prevalence in 1997 for the default scenarios was 3.1%, 7.8%, 28.9% and 27.1% in Cotonou, Yaoundé, Kisumu and Ndola, respectively, in line with data. Without biological cofactors, even implausibly high historical levels of risk behaviour in East Africa could not reproduce the observed heterogeneity in the late 1990s. Increasing the proportion of men circumcised in Ndola from 10% to 100% reduced HIV prevalence in 1997 to 7%. Decreasing the proportion circumcised in Yaoundé from 100% to 10% increased HIV prevalence to 26%.

Conclusions

Differences in male circumcision rates are likely to have played a key role in the heterogeneous spread of HIV across Africa. The effect of circumcision interventions can vary depending on the epidemic setting, with a larger effect in more generalised epidemics.
Differential modulation of right ventricular strain and right atrial mechanics in mild vs. severe pressure overload
Authors: Voeller Rochus K. , Aziz Abdulhameed , Maniar Hersh S. , Ufere Nneka N. , Taggar Ajay K. , Bernabe Noel J. , Cupps Brian P. , Moon Marc R.
Published: September 2011

Increased right atrial (RA) and ventricular (RV) chamber volumes are a late maladaptive response to chronic pulmonary hypertension. The purpose of the current investigation was to characterize the early compensatory changes that occur in the right heart during chronic RV pressure overload before the development of chamber dilation. Magnetic resonance imaging with radiofrequency tissue tagging was performed on dogs at baseline and after 10 wk of pulmonary artery banding to yield either mild RV pressure overload (36% rise in RV pressure; n = 5) or severe overload (250% rise in RV pressure; n = 4). The RV free wall was divided into three segments within a midventricular plane, and circumferential myocardial strain was calculated for each segment, the septum, and the left ventricle. Chamber volumes were calculated from stacked MRI images, and RA mechanics were characterized by calculating the RA reservoir, conduit, and pump contribution to RV filling. With mild RV overload, there were no changes in RV strain or RA function. With severe RV overload, RV circumferential strain diminished by 62% anterior ( P = 0.04), 42% inferior ( P = 0.03), and 50% in the septum ( P = 0.02), with no change in the left ventricle ( P = 0.12). RV filling became more dependent on RA conduit function, which increased from 30 ± 9 to 43 ± 13% ( P = 0.01), than on RA reservoir function, which decreased from 47 ± 6 to 33 ± 4% ( P = 0.04), with no change in RA pump function ( P = 0.94). RA and RV volumes and RV ejection fraction were unchanged from baseline during either mild ( P > 0.10) or severe RV pressure overload ( P > 0.53). In response to severe RV pressure overload, RV myocardial strain is segmentally diminished and RV filling becomes more dependent on RA conduit rather than reservoir function. These compensatory mechanisms of the right heart occur early in chronic RV pressure overload before chamber dilation develops.