bluuebunny/crossref_metadata_2025 huggingface dataset:
Multicopy Suppression Screening of Saccharomyces cerevisiae Identifies the Ubiquitination Machinery as a Main Target for Improving Growth at Low Temperatures
Authors: Hernández-López Maria José , García-Marqués Sara , Randez-Gil Francisca , Prieto Jose Antonio
Published: September 2011

A decrease in ambient temperature alters membrane functionality and impairs the proper interaction between the cell and its external milieu. Understanding how cells adapt membrane properties and modulate the activity of membrane-associated proteins is therefore of major interest from both the basic and the applied points of view. Here, we have isolated multicopy suppressors of the cold sensitivity phenotype of a
trp1
strain of
Saccharomyces cerevisiae
. Three poorly characterized genes, namely,
ALY2
encoding the endocytic adaptor,
CAJ1
encoding the J protein, and
UBP13
encoding the ubiquitin C-terminal hydrolase, were identified as mediating increased growth at 12°C of both Trp
−
and Trp
+
yeast strains. This effect was likely due to the downregulation of cold-instigated degradation of nutrient permeases, since it was missing from cells of the
rsp5
Δ mutant strain, which contains a point mutation in the gene encoding ubiquitin ligase. Indeed, we found that 12°C treatments reduced the level of several membrane transporters, including Tat1p and Tat2p, two yeast tryptophan transporters, and Gap1, the general amino acid permease. We also found that the lack of Rsp5p increased the steady state level of Tat1p and Tat2p and that
ALY2
-engineered cells grown at 12°C had higher Tat2p and Gap1p abundance. Nevertheless, the high copy number of
ALY2
or
UBP13
improved cold growth even in the absence of Tat2p. Consistent with this,
ALY2
- and
UBP13
-engineered cells of the industrial QA23 strain grew faster and produced more CO
2
at 12°C than did the parental when maltose was used as the sole carbon source. Hence, the multicopy suppressors isolated in this work appear to contribute to the correct control of the cell surface protein repertoire and their engineering might have potential biotechnological applications.
The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor
Authors: Santos-Beneit Fernando , Barriuso-Iglesias Mónica , Fernández-Martínez Lorena T. , Martínez-Castro Miriam , Sola-Landa Alberto , Rodríguez-García Antonio , Martín Juan F.
Published: September 2011

The RNA polymerase (RNAP) omega factor (ω) forms a complex with the α
2
ββ′ core of this enzyme in bacteria. We have characterized the
rpoZ
gene of
Streptomyces coelicolor
, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the
rpoZ
gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the Δ
rpoZ
strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the Δ
rpoZ
mutant with the wild-type
rpoZ
allele restored both phenotype and antibiotic production. Interestingly, the
rpoZ
gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that
rpoZ
promoter activity was increased in a Δ
phoP
background, it can be concluded that
rpoZ
is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in
Streptomyces
.
Molecular Epidemiology, Spatiotemporal Analysis, and Ecology of Sporadic Human Cryptosporidiosis in Australia
Authors: Waldron Liette S. , Dimeski Borce , Beggs Paul J. , Ferrari Belinda C. , Power Michelle L.
Published: September 2011

Parasites from the
Cryptosporidium
genus are the most common cause of waterborne disease around the world. Successful management and prevention of this emerging disease requires knowledge of the diversity of species causing human disease and their zoonotic sources. This study employed a spatiotemporal approach to investigate sporadic human cryptosporidiosis in New South Wales, Australia, between January 2008 and December 2010. Analysis of 261 human fecal samples showed that sporadic human cryptosporidiosis is caused by four species;
C. hominis
,
C. parvum
,
C. andersoni
, and
C. fayeri
. Sequence analysis of the
gp60
gene identified 5 subtype families and 31 subtypes.
Cryptosporidium hominis
IbA10G2 and
C. parvum
IIaA18G3R1 were the most frequent causes of human cryptosporidiosis in New South Wales, with 59% and 16% of infections, respectively, attributed to them. The results showed that infections were most prevalent in 0- to 4-year-olds. No gender bias or regional segregation was observed between the distribution of
C. hominis
and
C. parvum
infections. To determine the role of cattle in sporadic human infections in New South Wales, 205 cattle fecal samples were analyzed. Four
Cryptosporidium
species were identified,
C. hominis
,
C. parvum
,
C. bovis
, and
C. ryanae. C. parvum
subtype IIaA18G3R1 was the most common cause of cryptosporidiosis in cattle, with 47% of infections attributed to it.
C. hominis
subtype IbA10G2 was also identified in cattle isolates.
Molecular Epidemiology and Spatial Distribution of a Waterborne Cryptosporidiosis Outbreak in Australia
Authors: Waldron Liette S. , Ferrari Belinda C. , Cheung-Kwok-Sang Cristel , Beggs Paul J. , Stephens Nicola , Power Michelle L.
Published: September 2011

Cryptosporidiosis is one of the most common waterborne diseases reported worldwide. Outbreaks of this gastrointestinal disease, which is caused by the
Cryptosporidium
parasite, are often attributed to public swimming pools and municipal water supplies. Between the months of January and April in 2009, New South Wales, Australia, experienced the largest waterborne cryptosporidiosis outbreak reported in Australia to date. Through the course of the contamination event, 1,141 individuals became infected with
Cryptosporidium
. Health authorities in New South Wales indicated that public swimming pool use was a contributing factor in the outbreak. To identify the
Cryptosporidium
species responsible for the outbreak, fecal samples from infected patients were collected from hospitals and pathology companies throughout New South Wales for genetic analyses. Genetic characterization of
Cryptosporidium
oocysts from the fecal samples identified the anthroponotic
Cryptosporidium hominis
IbA10G2 subtype as the causative parasite. Equal proportions of infections were found in males and females, and an increased susceptibility was observed in the 0- to 4-year age group. Spatiotemporal analysis indicated that the outbreak was primarily confined to the densely populated coastal cities of Sydney and Newcastle.
Multilocus Fragment Typing and Genetic Structure of Cryptosporidium parvum Isolates from Diarrheic Preweaned Calves in Spain
Authors: Quílez Joaquín , Vergara-Castiblanco Claudia , Monteagudo Luis , del Cacho Emilio , Sánchez-Acedo Caridad
Published: September 2011

A collection of 140
Cryptosporidium parvum
isolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among the
C. parvum
isolates from cattle farms in this geographical area.
Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics
Authors: Jeon Jeong Ho , Kim Soo-Jin , Lee Hyun Sook , Cha Sun-Shin , Lee Jung Hun , Yoon Sang-Hong , Koo Bon-Sung , Lee Chang-Muk , Choi Sang Ho , Lee Sang Hee , Kang Sung Gyun , Lee Jung-Hyun
Published: September 2011

It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond ofp-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed inEscherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenicp-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for bothp-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds ofp-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.
Evidence for Direct Electron Transfer by a Gram-Positive Bacterium Isolated from a Microbial Fuel Cell
Authors: Wrighton K. C. , Thrash J. C. , Melnyk R. A. , Bigi J. P. , Byrne-Bailey K. G. , Remis J. P. , Schichnes D. , Auer M. , Chang C. J. , Coates J. D.
Published: September 2011

Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by
Thermincola potens
strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by
T. potens
strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that
c
-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction.
Salt Stress-Induced Changes in the Transcriptome, Compatible Solutes, and Membrane Lipids in the Facultatively Phototrophic Bacterium Rhodobacter sphaeroides
Authors: Tsuzuki Minoru , Moskvin Oleg V. , Kuribayashi Masayuki , Sato Kiichi , Retamal Susana , Abo Mitsuru , Zeilstra-Ryalls Jill , Gomelsky Mark
Published: September 2011

Responses to NaCl stress were investigated in phototrophically grown
Alphaproteobacterium Rhodobacter sphaeroides
by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems,
proVWX
and
betS
, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in
R. sphaeroides
, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon
otsBA
. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors,
crpO
(RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When
crpO
was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in
R. sphaeroides
.
Formation of Polyhydroxyalkanoate in Aerobic Anoxygenic Phototrophic Bacteria and Its Relationship to Carbon Source and Light Availability
Authors: Xiao Na , Jiao Nianzhi
Published: September 2011

Aerobic anoxygenic phototrophic bacteria (AAPB) are unique players in carbon cycling in the ocean. Cellular carbon storage is an important mechanism regulating the nutrition status of AAPB but is not yet well understood. In this paper, six AAPB species (
Dinoroseobacter
sp. JL1447,
Roseobacter denitrificans
OCh 114,
Roseobacter litoralis
OCh 149,
Dinoroseobacter shibae
DFL 12
T
,
Labrenzia alexandrii
DFL 11
T
, and
Erythrobacter longus
DSMZ 6997) were examined, and all of them demonstrated the ability to form the carbon polymer polyhydroxyalkanoate (PHA) in the cell. The PHA in
Dinoroseobacter
sp. JL1447 was identified as poly-beta-hydroxybutyrate (PHB) according to evidence from Fourier transform infrared spectroscopy, differential scanning calorimetry, and
1
H nuclear magnetic resonance spectroscopy examinations. Carbon sources turned out to be critical for PHA production in AAPB. Among the eight media tested with
Dinoroseobacter
sp. JL1447, sodium acetate, giving a PHA production rate of 72%, was the most productive carbon source, followed by glucose, with a 68% PHA production rate. Such PHA production rates are among the highest recorded for all bacteria. The C/N ratio of substrates was verified by the experiments as another key factor in PHA production. In the case of
R. denitrificans
OCh 114, PHA was not detected when the organism was cultured at C/N ratios of <2 but became apparent at C/N ratios of >3. Light is also important for the formation of PHA in AAPB. In the case of
Dinoroseobacter
sp. JL1447, up to a one-quarter increase in PHB production was observed when the culture underwent growth in a light-dark cycle compared to growth completely in the dark.
Partial Genome Assembly for a Candidate Division OP11 Single Cell from an Anoxic Spring (Zodletone Spring, Oklahoma)
Authors: Youssef Noha H. , Blainey Paul C. , Quake Stephen R. , Elshahed Mostafa S.
Published: September 2011

Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.
Vacuolar Protein Sorting Genes Regulate Mat Formation in Saccharomyces cerevisiae by Flo11p-Dependent and -Independent Mechanisms
Authors: Sarode Neha , Miracle Bethany , Peng Xin , Ryan Owen , Reynolds Todd B.
Published: September 2011

Saccharomyces cerevisiae
generates complex biofilms called mats on low-density (0.3%) agar plates. The mats can be morphologically divided into two regions: (i) hub, the interior region characterized by the presence of wrinkles and channels, and (ii) rim, the smooth periphery. Formation of mats depends on the adhesin Flo11p, which is also required for invasive growth, a phenotype in which the
S. cerevisiae
yeasts grow as chains of cells that dig into standard-density (2%) agar plates. Although both invasive growth and mat formation depend on Flo11p, mutations that perturb the multivesicular body (MVB) protein sorting pathway inhibit mat formation in a
FLO11
-independent manner. These mutants, represented by
vps27
Δ, disrupt mat formation but do not affect invasive growth,
FLO11
gene or protein expression, or Flo11p localization. In contrast, an overlapping subset of MVB mutants (represented by ESCRT [endosomal sorting complex required for transport] complex genes such as
VPS25
) interrupt the Rim101p signal transduction cascade, which is required for
FLO11
expression, and thus block both invasive growth and mat formation. In addition, this report shows that mature Flo11p is covalently associated with the cell wall and shed into the extracellular matrix of the growing mat.
A GCN2-Like Eukaryotic Initiation Factor 2 Kinase Increases the Viability of Extracellular Toxoplasma gondii Parasites
Authors: Konrad Christian , Wek Ronald C. , Sullivan William J.
Published: September 2011

Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite
Toxoplasma gondii
, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation.
Toxoplasma
parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of
Toxoplasma
eukaryotic translation initiation factor 2-alpha (TgIF2α) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2α kinase designated TgIF2K-D that is related to GCN2, a eukaryotic initiation factor 2α (eIF2α) kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular
Toxoplasma
parasites and facilitates translational control through TgIF2α phosphorylation in extracellular parasites. We generated a
TgIF2K-D
knockout parasite and demonstrated that loss of this eIF2α kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside host cells. This phenotype is consistent with that reported for our nonphosphorylatable TgIF2α mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2α kinase responsible for promoting extracellular viability of
Toxoplasma
. These studies suggest that eIF2α phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2α is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K.
Sex Determination in the First-Described Sexual Fungus
Authors: Idnurm Alexander
Published: September 2011

The original report of sex in fungi dates 2 centuries ago to the species
Syzygites megalocarpus
(Mucoromycotina). The organism was subsequently used in 1904 to represent self-fertile homothallic species when the concepts of heterothallism and homothallism were developed for the fungal kingdom. In this study, two putative
sex
/
MAT
loci were identified in individual strains of
S. megalocarpus
, accounting for its homothallic behavior. The strains encode both of the high-mobility-group domain-containing proteins, SexM and SexP, flanked by RNA helicase and glutathione oxidoreductase genes that are found adjacent to the mating-type loci in other Mucoromycotina species. The presence of pseudogenes and the arrangement of genes suggest that the origin of homothallism in this species is from a heterothallic relative, obtained via a chromosomal rearrangement to switch two alleles into two separated loci within a single genetic background. Similar events have given rise to homothallic species from heterothallic species in ascomycete fungi, demonstrating that conserved forces shape the evolution of sex determination and speciation in highly diverged fungi.
Diversity in Requirement of Genetic and Epigenetic Factors for Centromere Function in Fungi
Authors: Roy Babhrubahan , Sanyal Kaustuv
Published: September 2011

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for
de novo
centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species.
Sampangine Inhibits Heme Biosynthesis in both Yeast and Human
Authors: Huang Zhiwei , Chen Kaifu , Xu Tao , Zhang Jianhuai , Li Yongxiang , Li Wei , Agarwal Ameeta K. , Clark Alice M. , Phillips John D. , Pan Xuewen
Published: September 2011

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast
Saccharomyces cerevisiae
. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism—by hyperactivating uroporhyrinogen III synthase—to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.
An Erythrocyte Cytoskeleton-Binding Motif in Exported Plasmodium falciparum Proteins
Authors: Kilili Geoffrey K. , LaCount Douglas J.
Published: September 2011

Binding of exported malaria parasite proteins to the host cell membrane and cytoskeleton contributes to the morphological, functional, and antigenic changes seen in
Plasmodium falciparum
-infected erythrocytes. One such exported protein that targets the erythrocyte cytoskeleton is the mature parasite-infected erythrocyte surface antigen (MESA), which interacts with the N-terminal 30-kDa domain of protein 4.1R via a 19-residue sequence. We report here that the MESA erythrocyte cytoskeleton-binding (MEC) domain is present in at least 13 other
P. falciparum
proteins predicted to be exported to the host cell. An alignment of the putative cytoskeleton-binding sequences revealed a conserved aspartic acid at the C terminus that was omitted from the originally reported binding domain. Mutagenesis experiments demonstrated that this aspartic acid was required for the optimal binding of MESA to inside-out vesicles (IOVs) prepared from erythrocytes. Using pulldown assays, we characterized the binding of fragments encoding the MEC domains from PFE0040c/MESA and six other proteins (PF10_0378, PFA0675w, PFB0925w, PFD0095c, PFF1510w, and PFI1790w) to IOVs. All seven proteins bound to IOVs, with MESA showing the strongest affinity in saturation binding experiments. We further examined the interaction of the MEC domain proteins with components of the erythrocyte cytoskeleton and showed that MESA, PF10_0378, and PFA0675w coprecipitated full-length 4.1R from lysates prepared from IOVs. These data demonstrated that the MEC motif is present and functional in at least six other
P. falciparum
proteins that are exported to the host cell cytoplasm.
Regulation of
perR
Expression by Iron and PerR in Campylobacter jejuni
Authors: Kim Minkyeong , Hwang Sunyoung , Ryu Sangryeol , Jeon Byeonghwa
Published: September 2011

Campylobacter jejuni
is a leading food-borne pathogen causing gastroenteritis in humans. Although OxyR is a widespread oxidative stress regulator in many Gram-negative bacteria,
C. jejuni
lacks OxyR and instead possesses the metalloregulator PerR. Despite the important role played by PerR in oxidative stress defense, little is known about the factors influencing
perR
expression in
C. jejuni
. In this study, a
perR
promoter-
lacZ
fusion assay demonstrated that iron significantly reduced the level of
perR
transcription, whereas other metal ions, such as copper, cobalt, manganese, and zinc, did not affect
perR
transcription. Notably, a
perR
mutation substantially increased the level of
perR
transcription and in
trans
complementation restored the transcriptional changes, suggesting
perR
is transcriptionally autoregulated in
C. jejuni
. In the
perR
mutant, iron did not repress
perR
transcription, indicating the iron dependence of
perR
expression results from
perR
autoregulation. Electrophoretic mobility shift assays showed that PerR binds to the
perR
promoter, and DNase I footprinting assays identified a PerR binding site overlapping the −35 region of the two
perR
promoters, further supporting
perR
autoregulation at the transcriptional level. Alignment of the PerR binding sequence in the
perR
promoter with the regulatory region of other PerR regulon genes of
C. jejuni
revealed a 16-bp consensus PerR binding sequence, which shares high similarities to the
Bacillus subtilis
PerR box. The results of this study demonstrated that PerR directly interacts with the
perR
promoter and regulates
perR
transcription and that
perR
autoregulation is responsible for the repression of
perR
transcription by iron in
C. jejuni
.
Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems
Authors: Makarova Kira S. , Wolf Yuri I. , Snir Sagi , Koonin Eugene V.
Published: September 2011

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.
Recognition of β-Strand Motifs by RseB Is Required for σ
E
Activity in Escherichia coli
Authors: Kulp Adam , Kuehn Meta J.
Published: September 2011

Gram-negative bacteria react to misfolded proteins in the envelope through a myriad of different stress response pathways. This cohort of pathways allows the bacteria to specifically respond to different types of damage, and many of these have been discovered to have key roles in the virulence of bacterial pathogens. Misfolded outer membrane proteins (OMPs) are typically recognized by the σ
E
pathway, a highly conserved envelope stress response pathway. We examined the features of misfolded OMPs with respect to their ability to generate envelope stress responses. We determined that the secondary structure, particularly the potential to form β strands, is critical to inducing the σ
E
response in an RseB-dependent manner. The sequence of the potential β-strand motif modulates the strength of the σ
E
response generated by the constructs. By understanding the details of how such stress response pathways are activated, we can gain a greater understanding of how bacteria survive in harsh environments.
Determination of Borrelia Surface Lipoprotein Anchor Topology by Surface Proteolysis
Authors: Chen Shiyong , Kumru Ozan S. , Zückert Wolfram R.
Published: September 2011

We used a surface trypsinolysis assay to probe accessibility of the membrane-proximal N-terminal tether peptides of
Borrelia
surface lipoproteins OspA and Vsp1. Our findings with both wild-type and mutant proteins are only compatible with the anchoring of these surface lipoproteins in the outer leaflet of the outer spirochetal membrane.
An Atypical Phr Peptide Regulates the Developmental Switch Protein RapH
Authors: Mirouze Nicolas , Parashar Vijay , Baker Melinda D. , Dubnau David A. , Neiditch Matthew B.
Published: September 2011

Under conditions of nutrient limitation and high population density, the bacterium
Bacillus subtilis
can initiate a variety of developmental pathways. The signaling systems regulating
B. subtilis
differentiation are tightly controlled by switch proteins called Raps, named after the founding members of the family, which were shown to be
r
esponse regulator
a
spartate
p
hosphatases. A
phr
gene encoding a secreted pentapeptide that regulates the activity of its associated Rap protein was previously identified downstream of 8 of the chromosomally encoded
rap
genes. We identify and validate here the sequence of an atypical Phr peptide, PhrH, by
in vivo
and
in vitro
analyses. Using a luciferase reporter bioassay combined with
in vitro
experiments, we found that PhrH is a hexapeptide (TDRNTT), in contrast to the other characterized Phr pentapeptides. We also determined that
phrH
expression is driven by a promoter lying within
rapH
. Unlike the previously identified dedicated σ
H
-driven
phr
promoters, it appears that
phrH
expression most likely requires σ
A
. Furthermore, we show that PhrH can antagonize both of the known activities of RapH: the dephosphorylation of Spo0F and the sequestration of ComA, thus promoting the development of spores and the competent state. Finally, we propose that PhrH is the prototype of a newly identified class of Phr signaling molecules consisting of six amino acids. This class likely includes PhrI, which regulates RapI and the expression, excision, and transfer of the mobile genetic element ICE
Bs
1.
The Escherichia coli MntR Miniregulon Includes Genes Encoding a Small Protein and an Efflux Pump Required for Manganese Homeostasis
Authors: Waters Lauren S. , Sandoval Melissa , Storz Gisela
Published: September 2011

Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only
Escherichia coli
K-12 gene known to respond to manganese via MntR repression was
mntH
, which encodes a manganese importer. We show that
mntS
(formerly the small RNA gene
rybA
) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of
mntH
. We also provide evidence that
mntP
(formerly
yebN
), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of
mntP
leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action.
Transfer of R388 Derivatives by a Pathogenesis-Associated Type IV Secretion System into both Bacteria and Human Cells
Authors: Fernández-González Esther , de Paz Héctor D. , Alperi Anabel , Agúndez Leticia , Faustmann Marco , Sangari Félix J. , Dehio Christoph , Llosa Matxalen
Published: September 2011

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen
Bartonella henselae
to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies.
B. henselae
codes for two T4SSs: VirB/VirD4 and Trw. A Δ
virB
mutant strain was transfer deficient, while a Δ
trwE
mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels.
B. henselae
bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient
Escherichia coli
under certain conditions, underscoring the versatility of T4SSs.
The Transcript from the σ
28
-Dependent Promoter Is Translationally Inert in the Expression of the σ
28
-Encoding Gene
fliA
in the
fliAZ
Operon of Salmonella enterica Serovar Typhimurium
Authors: Tanabe Yasushi , Wada Takeo , Ono Katsuhiko , Abo Tatsuhiko , Kutsukake Kazuhiro
Published: September 2011

There are three classes of promoters for flagellar operons in
Salmonella
. Class 2 promoters are transcribed by σ
70
RNA polymerase in the presence of an essential activator, FlhD
4
C
2
, and activated by an auxiliary regulator, FliZ. Class 3 promoters are transcribed by σ
28
RNA polymerase and repressed by an anti-σ
28
factor, FlgM. σ
28
(FliA) and FliZ are encoded by the
fliA
and
fliZ
genes, respectively, which together constitute an operon transcribed in this order. This operon is transcribed from both class 2 and class 3 promoters, suggesting that it should be activated by its own product, σ
28
, even in the absence of FlhD
4
C
2
. However, σ
28
-dependent transcription occurs
in vivo
only in the presence of FlhD
4
C
2
, indicating that transcription from the class 2 promoter is a prerequisite to that from the class 3 promoter. In this study, we examined the effects of variously modified versions of the
fliA
regulatory region on transcription and translation of the
fliA
gene. We showed that FliA is not significantly translated from the class 3 transcript. In contrast, the 5′-terminal AU-rich sequence found in the class 2 transcript confers efficient
fliA
translation. Replacement of the Shine-Dalgarno sequence of the
fliA
gene with a better one improved
fliA
translation from the class 3 transcript. These results suggest that the 5′-terminal AU-rich sequence of the class 2 transcript may assist ribosome binding. FliZ was shown to be expressed from both the class 2 and class 3 transcripts.
Improved clinical outcomes and efficacy with a nurse-led colonoscopy surveillance service
Authors: Sarkar Sanchoy , Duffy Una , Haslam Neil
Published: September 2011

Background

A nurse practitioner-led colonoscopy surveillance service was introduced to improve appropriateness, validation and compliance with the National Patient Safety Agency safety alert and British Society of Gastroenterology consensus guidance for bowel cleansing agents.

Objective

To determine the clinical outcomes and efficacy of this new service.

Design and patients

A 4-month prospective audit of patients due to attend for surveillance colonoscopy.

Setting

Royal Liverpool University Hospital.

Intervention

A new nurse practitioner-led surveillance service reviewed all patients before listing.

Outcomes

Clinical outcomes, service efficiency and cost effectiveness.

Results

224 Patients (median age 68 years, 52% male, and median American Society of Anesthesiologists (ASA) 2) were assessed and 34% had medical factors influencing their colonoscopy. 37% patients were discharged without a colonoscopy, 17% deferred (median >2 years), 6% had died while on the register and the remaining (40%) had their procedure at the agreed interval. The 30-day and 6-month all-cause mortality was 0% for those fit for colonoscopy, compared with 5% and 14%, respectively, for those deemed unfit. The did-not-attend (DNA) rate was reduced from 7.6% to <1%. With 95 patients not requiring a colonoscopy a potential £40 000 saving to the primary care trust was made.

Conclusions

The nurse practitioner-led surveillance service has been invaluable for guideline adherence and medical management of patients before colonoscopy. In addition, it potentially avoided procedural all-cause mortality in these patients. It has proved to be efficacious with reduced DNA rates and over one-third of patients assessed did not require a colonoscopy.
Use of diagnostic markers to monitor fasciolosis and gastrointestinal nematodes on an organic dairy farm
Authors: Ellis K. A. , Jackson A. , Bexiga R. , Matthews J. , McGoldrick J. , Gilleard J. , Forbes A. B.
Published: September 2011

A 12‐month study was conducted to assess and monitor gastrointestinal tract nematodes and liver fluke in cohorts of cattle on a Scottish organic dairy farm. Various diagnostic markers for helminth parasites of cattle from different age groups were assessed monthly from April 2007 to March 2008. First season grazing stock were subjected to significant challenge fromOstertagia ostertaginematodes as reflected in serum pepsinogen concentrations, which rose markedly in the second half of the grazing season. In addition, plasma albumin concentrations decreased and faecal egg counts (FEC) increased moderately, indicating exposure to bothO ostertagiand probablyCooperia oncophora. Second season grazing animals had a peak FEC early in the grazing period, suggestive of a potential carry‐over ofOstertagiaspecies infection (‘Type 2’) during housing. All classes of cattle showed evidence of fluke (Fasciola hepatica) infection. Adult cow exposure toO ostertagiand fluke was estimated via the use of ELISA testing to detect antibodies toO ostertagiandF hepaticaand the high levels detected suggested a significant exposure response. Despite low stocking densities and sympathetic grazing management, there was a significant challenge to all grazing stock from gastrointestinal nematodes and liver fluke.
Effect of personal response systems on student perception and academic performance in courses in a health sciences curriculum
Authors: FitzPatrick Kathleen A. , Finn Kevin E. , Campisi Jay
Published: September 2011

To increase student engagement, active participation, and performance, personal response systems (clickers) were incorporated into six lecture-based sections of four required courses within the Health Sciences Department major curriculum: freshman-level Anatomy and Physiology I and II, junior-level Exercise Physiology, and senior-level Human Pathophysiology. Clickers were used to gather anonymous student responses to questions posed within the class period after individual thought and peer discussion. Students ( n = 293, 88% of students completing the courses) completed a perceptual survey on clicker effectiveness inserted into the Student Assessment of Learning Gains online instrument. Across courses and years, students uniformly rated several dimensions of clicker use as providing good to great gain in engaging them in active learning, increasing participation and involvement during class, maintaining attention, applying material immediately, providing feedback concerning their understanding, and offering an anonymous format for participation. Within these four sections, quiz grades were compared between clicker and nonclicker years. Significant increases in pre- and posttest scores were seen in Exercise Physiology in clicker years and on some, but not all material, in Anatomy and Physiology I and II based on content quizzes. Human Pathophysiology results were unexpected, with higher quiz scores in the nonclicker year. The results support the hypothesis of increased engagement with clicker use. The hypothesis of increased student performance was not consistently supported. Increased performance was seen in Exercise Physiology. In Anatomy and Physiology I and II, performance improved on some content quizzes. In Human Pathophysiology, performance did not improve with clickers.
A Web-based course of lectures in respiratory physiology
Authors: West John B.
Published: September 2011

A complete course of respiratory physiology suitable for first-year medical and graduate students has been placed on the Web for our own students and for other educational institutions. There are several reasons for doing this. The first is that the modern-day student uses a variety of options for acquiring knowledge. These include attending lectures, reading texts, iPod downloads, and surfing the internet. This Web-based course is another option that may be preferable for some students. A second reason is that it is becoming increasingly difficult for some medical schools to find faculty members who are willing and able to teach the principles of respiratory physiology. This is a potentially serious problem because a sound knowledge of respiratory physiology will always be necessary for the intelligent practice of medicine. Schools with limited faculty may find it useful to use these Web-based lectures followed by a discussion session with students. Another reason is that some schools have moved away from systematic lectures to case-based discussions, with the possibility that students will not be exposed to some of the principles of respiratory physiology. The hope is that this comprehensive course of lectures will help students to assimilate this important material as the medical school curriculum continues to expand at a rapid rate.