bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of the Chiral Compound (
R
)-3-Hydroxybutyrate by a Genetically Engineered Methylotrophic Bacterium
Authors: Hölscher Tina , Breuer Uta , Adrian Lorenz , Harms Hauke , Maskow Thomas 
Published: June 2010

ABSTRACT

In this study, a methylotrophic bacterium,
Methylobacterium rhodesianum
MB 126, was used for the production of the chiral compound (
R
)-3-hydroxybutyrate (
R
-3HB) from methanol.
R
-3HB is formed during intracellular degradation of the storage polymer (
R
)-3-polyhydroxybutyrate (PHB). Since the monomer
R
-3HB does not accumulate under natural conditions,
M. rhodesianum
was genetically modified. The gene (
hbd
) encoding the
R
-3HB-degrading enzyme,
R
-3HB dehydrogenase, was inactivated in
M. rhodesianum
. The resulting
hbd
mutant still exhibited low growth rates on
R
-3HB as the sole source of carbon and energy, indicating the presence of alternative pathways for
R
-3HB utilization. Therefore, transposon mutagenesis was carried out with the
hbd
mutant, and a double mutant unable to grow on
R
-3HB was obtained. This mutant was shown to be defective in lipoic acid synthase (LipA), resulting in an incomplete citric acid cycle. Using the
hbd lipA
mutant, we produced 3.2 to 3.5 mM
R
-3HB in batch and 27 mM (2,800 mg liter
−1
) in fed-batch cultures. This was achieved by sequences of cultivation conditions initially favoring growth, then PHB accumulation, and finally PHB degradation.
Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in
Saccharomyces cerevisiae
Authors: Zelle Rintze M. , Trueheart Josh , Harrison Jacob C. , Pronk Jack T. , van Maris Antonius J. A. 
Published: June 2010

ABSTRACT

Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type
Saccharomyces cerevisiae
. Pyruvate carboxylase-negative (Pyc
−
)
S. cerevisiae
strains cannot grow on glucose unless media are supplemented with C
4
compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the
S. cerevisiae
PEPCK encoded by
PCK1
is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in
S. cerevisiae
. Pyc
−
S. cerevisiae
strains constitutively overexpressing the PEPCK either from
S. cerevisiae
or from
Actinobacillus succinogenes
did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h
−1
, one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C
4
dicarboxylic acids in
S. cerevisiae
.
Freshwater Suspended Sediments and Sewage Are Reservoirs for Enterotoxin-Positive
Clostridium perfringens
Authors: Mueller-Spitz Sabrina R. , Stewart Lisa B. , Klump J. Val , McLellan Sandra L. 
Published: June 2010

ABSTRACT

The release of fecal pollution into surface waters may create environmental reservoirs of feces-derived microorganisms, including pathogens.
Clostridium perfringens
is a commonly used fecal indicator that represents a human pathogen. The pathogenicity of this bacterium is associated with its expression of multiple toxins; however, the prevalence of
C. perfringens
with various toxin genes in aquatic environments is not well characterized. In this study,
C. perfringens
spores were used to measure the distribution of fecal pollution associated with suspended sediments in the nearshore waters of Lake Michigan. Particle-associated
C. perfringens
levels were greatest adjacent to the Milwaukee harbor and diminished in the nearshore waters. Species-specific PCR and toxin gene profiles identified 174 isolates collected from the suspended sediments, surface water, and sewage influent as
C. perfringens
type A. Regardless of the isolation source, the beta2 and enterotoxin genes were common among isolates. The suspended sediments yielded the highest frequency of
cpe
-carrying
C. perfringens
(61%) compared to sewage (38%). Gene arrangement of enterotoxin was investigated using PCR to target known insertion sequences associated with this gene. Amplification products were detected in only 9 of 90 strains, which suggests there is greater variability in
cpe
gene arrangement than previously described. This work presents evidence that freshwater suspended sediments and sewage influent are reservoirs for potentially pathogenic
cpe
-carrying
C. perfringens
spores.
Detection of
Vibrio cholerae
O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay
Authors: Wang Duochun , Xu Xuebin , Deng Xiaoling , Chen Changyi , Li Baisheng , Tan Hailing , Wang Haibo , Tang Song , Qiu Haiyan , Chen Jingdiao , Ke Bixia , Ke Changwen , Kan Biao 
Published: June 2010

ABSTRACT

Environmental waters are an important reservoir for
Vibrio cholerae
, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect
V. cholerae
O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 10
3
CFU/ml for detection of
V. cholerae
O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 10
5
CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the
V. cholerae
O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146;
P
< 0.01) but lower than that of real-time PCR (29.5%, 43/146;
P
< 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the
V. cholerae
concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of
V. cholerae
in environmental water samples.
Molecular and Genetic Analyses of the Putative
Proteus
O Antigen Gene Locus
Authors: Wang Quan , Torzewska Agnieszka , Ruan Xiaojuan , Wang Xiaoting , Rozalski Antoni , Shao Zhujun , Guo Xi , Zhou Haijian , Feng Lu , Wang Lei 
Published: June 2010

ABSTRACT

Proteus
species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The
Proteus
O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of
Proteus
isolates. Even though most
Proteus
O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative
Proteus
O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40
Proteus
isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative
Proteus
O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally,
Proteus
-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68
Proteus
O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative
Proteus
O antigen locus, and we describe a novel molecular classification method for the identification of different
Proteus
serogroups.
Two Functionally Distinct Ciliates Dwelling in
Acropora
Corals in the South China Sea near Sanya, Hainan Province, China
Authors: Qiu Dajun , Huang Liangmin , Huang Hui , Yang Jianhui , Lin Senjie 
Published: June 2010

ABSTRACT

We detected and characterized two distinct scuticociliate ciliates inside
Acropora
corals in the South China Sea. One, voraciously foraging on
Symbiodinium
, resembled the brown band disease of ciliates. The other, which is closely related to
Paranophrys magna
, grazed on detritus instead of
Symbiodinium
. These two ciliates may serve contrasting functions (competitor versus “cleaner”) in the coral-ciliate-
Symbiodinium
triangular relationship.
Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs
Authors: Warnes S. L. , Green S. M. , Michels H. T. , Keevil C. W. 
Published: June 2010

ABSTRACT

The increasing incidence of nosocomial infections caused by glycopeptide-resistant enterococci is a global concern. Enterococcal species are also difficult to eradicate with existing cleaning regimens; they can survive for long periods on surfaces, thus contributing to cases of reinfection and spread of antibiotic-resistant strains. We have investigated the potential use of copper alloys as bactericidal surfaces. Clinical isolates of vancomycin-resistant
Enterococcus faecalis
and
Enterococcus faecium
were inoculated onto copper alloy and stainless steel surfaces. Samples were assessed for the presence of viable cells by conventional culture, detection of actively respiring cells, and assessment of cell membrane integrity. Both species survived for up to several weeks on stainless steel. However, no viable cells were detected on any alloys following exposure for 1 h at an inoculum concentration of ≤10
4
CFU/cm
2
. Analysis of genomic and plasmid DNA from bacterial cells recovered from metal surfaces indicates substantial disintegration of the DNA following exposure to copper surfaces that is not evident in cells recovered from stainless steel. The DNA fragmentation is so extensive, and coupled with the rapid cell death which occurs on copper surfaces, that it suggests that mutation is less likely to occur. It is therefore highly unlikely that genetic information can be transferred to receptive organisms recontaminating the same area. A combination of effective cleaning regimens and contact surfaces containing copper could be useful not only to prevent the spread of viable pathogenic enterococci but also to mitigate against the occurrence of potential resistance to copper, biocides, or antibiotics and the spread of genetic determinants of resistance to other species.
C Terminus of Nce102 Determines the Structure and Function of Microdomains in the Saccharomyces cerevisiae Plasma Membrane
Authors: Loibl Martin , Grossmann Guido , Stradalova Vendula , Klingl Andreas , Rachel Reinhard , Tanner Widmar , Malinsky Jan , Opekarová Miroslava 
Published: June 2010

ABSTRACT

The plasma membrane of the yeast
Saccharomyces cerevisiae
contains stably distributed lateral domains of specific composition and structure, termed MCC (
m
embrane
c
ompartment of arginine permease
C
an1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an
NCE102
deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the
NCE102
close homolog
FHN1
(the previously uncharacterized
YGR131W
) as well as by distant
Schizosaccharomyces pombe
homolog
fhn1
(
SPBC1685.13
). We conclude that this mechanism of plasma membrane organization is conserved through the phylum
Ascomycota
. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.
Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis
Authors: Baxt Leigh A. , Rastew Elena , Bracha Rivka , Mirelman David , Singh Upinder 
Published: June 2010

ABSTRACTEntamoeba histolyticais a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown thatE. histolyticacontains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.
The Candida albicans ESCRT Pathway Makes Rim101-Dependent and -Independent Contributions to Pathogenesis
Authors: Wolf Julie M. , Johnson Diedre J. , Chmielewski David , Davis Dana A. 
Published: June 2010

ABSTRACT

Candida albicans
is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environmental characteristics. To adapt to these different sites,
C. albicans
must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subsequent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the
vps27
Δ/Δ mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the
vps27
Δ/Δ strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for
C. albicans
virulence.
Cryptococcus neoformans Histone Acetyltransferase Gcn5 Regulates Fungal Adaptation to the Host
Authors: O'Meara Teresa R. , Hay Christie , Price Michael S. , Giles Steve , Alspaugh J. Andrew 
Published: June 2010

ABSTRACT

Cryptococcus neoformans
is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses, we identified the
C. neoformans
Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of
Saccharomyces cerevisiae
. The
C. neoformans
gcn5
Δ mutant strain has defects in high-temperature growth and capsule attachment to the cell surface, in addition to increased sensitivity to FK506 and oxidative stress. Treatment of wild-type cells with the histone acetyltransferase inhibitor garcinol mimics cellular effects of the
gcn5
Δ
mutation. Gcn5 regulates the expression of many genes that are important in responding to the specific environmental conditions encountered by
C. neoformans
inside the host. Accordingly, the
gcn5
Δ
mutant is avirulent in animal models of cryptococcosis. Our study demonstrates the importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow
C. neoformans
to respond appropriately to the human host.
The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?
Authors: Günzl Arthur 
Published: June 2010

ABSTRACT

Trypanosomatids are early-diverged, protistan parasites of which
Trypanosoma brucei
,
Trypanosoma cruzi
, and several species of
Leishmania
cause severe, often lethal diseases in humans. To better combat these parasites, their molecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL)
trans
splicing in
T. brucei
established a key difference between parasites and hosts. In SL
trans
splicing, the capped 5′-terminal region of the small nuclear SL RNA is fused onto the 5′ end of each mRNA. This process, in conjunction with polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA by providing the cap structure. The reaction is a two-step transesterification process analogous to intron removal by
cis
splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to ∼170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicing factors were identified by standard genome annotation because trypanosomatid amino acid sequences are among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterization of the splicing factor repertoire in
T. brucei
, achieved by tandem affinity purification of splicing complexes, by systematic analysis of proteins containing RNA recognition motifs, and by mining the genome database. In addition, recent findings about functional differences between trypanosome and human pre-mRNA splicing factors are discussed.
Uga3 and Uga35/Dal81 Transcription Factors Regulate
UGA4
Transcription in Response to γ-Aminobutyric Acid and Leucine
Authors: Cardillo Sabrina Beatriz , Bermúdez Moretti Mariana , Correa García Susana 
Published: June 2010

ABSTRACT

The
Saccharomyces cerevisiae
UGA4
gene encodes a permease capable of importing γ-aminobutyric acid (GABA) and δ-aminolevulinic acid (ALA) into the cell. GABA-dependent induction of this permease requires at least two positive-acting proteins, the specific factor Uga3 and the pleiotropic factor Uga35/Dal81.
UGA4
is subjected to a very complex regulation, and its induction is affected by the presence of extracellular amino acids; this effect is mediated by the plasma membrane amino acid sensor SPS. Our results show that leucine affects
UGA4
induction and that the SPS sensor and the downstream effectors Stp1 and Stp2 participate in this regulation. Moreover, we found that the Uga3 and Uga35/Dal81 transcription factors bind to the
UGA4
promoter in a GABA-dependent manner and that this binding is impaired by the presence of leucine. We also found that the Leu3 transcription factor negatively regulates
UGA4
transcription, although this seems to be through an indirect mechanism.
Characterization of N-Linked Protein Glycosylation in
Helicobacter pullorum
Authors: Jervis Adrian J. , Langdon Rebecca , Hitchen Paul , Lawson Andrew J. , Wood Alison , Fothergill Joanne L. , Morris Howard R. , Dell Anne , Wren Brendan , Linton Dennis 
Published: June 2010

ABSTRACT

The first bacterial N-linked glycosylation system was discovered in
Campylobacter jejuni
, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that
pglB
orthologues are present in a subset of species from the
Deltaproteobacteria
and
Epsilonproteobacteria
, including three
Helicobacter
species:
H. pullorum
,
H. canadensis
, and
H. winghamensis
. In contrast to
C. jejuni
, in which a single
pglB
gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these
Helicobacter
species contain two unrelated
pglB
genes (
pglB1
and
pglB2
), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the
H. pullorum
PglB1 protein, but not PglB2, was able to transfer
C. jejuni
N-linked glycan onto an acceptor protein in
Escherichia coli
. Analysis of the characterized
C. jejuni
N-glycosylation system with an
in vitro
oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to
H. pullorum
, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the −2 position of the N-glycosylation sequon, as for
C. jejuni
. Attempted insertional knockout mutagenesis of the
H. pullorum pglB2
gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied
C. jejuni
system.