bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Characterization of a New Family 45 Endoglucanase, STCE1, from
Staphylotrichum coccosporum
and Its Overproduction in
Humicola insolens
Authors: Koga Jinichiro , Baba Yuko , Shimonaka Atsushi , Nishimura Tomoko , Hanamura Satoshi , Kono Toshiaki 
Published: April 2008

ABSTRACT

In the detergent industry, fungal endoglucanases have been used to release microfibrils (defibrillation) from the surface of dyed cellulosic fabrics to enhance color brightness. Although endoglucanases for laundry use must have various properties, such as a neutral or alkaline optimum pH, resistance to anionic surfactants and oxidizing agents (main components in detergents), and high defibrillation activity, all-purpose endoglucanases have not been obtained yet. As a result of screening of endoglucanases, a new family 45 endoglucanase (family 45 glycoside hydrolase), designated STCE1, was obtained and purified to apparent homogeneity from the culture supernatant of
Staphylotrichum coccosporum
NBRC 31817. The molecular mass of STCE1 was 49 kDa. The optimum pH for the carboxymethyl cellulase activity of STCE1 was 6.0, and the optimum temperature was 60°C. STCE1 was highly resistant to an anionic surfactant and an oxidizing agent. Furthermore, the defibrillation activities on dyed cotton and lyocell fabrics of STCE1 were higher than those of the other representative endoglucanases tested. These results indicate that STCE1 is an all-purpose enzyme for laundry use. A gene encoding STCE1, designated the
stce1
gene, was cloned from
S. coccosporum
, and the complete sequence was determined. STCE1 consisted of three distinct domains: an N-terminal catalytic domain (family 45), a linker domain, and a C-terminal carbohydrate-binding module (family 1). The amino acid sequences of the catalytic domain of STCE1 were phylogenetically close to those of the family 45 endoglucanases EGL3, EGL4, and EGV from a
Humicola
sp. Hence, the
stce1
gene was transferred into
Humicola insolens
and expressed. As a result, extremely high levels (0.90 mg protein per ml of culture supernatant, 27% of the total proteins) of the recombinant STCE1 were secreted as a mature form in the culture supernatant.
Investigating the Properties of
Bacillus thuringiensis
Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology
Authors: Pigott Craig R. , King Martin S. , Ellar David J. 
Published: April 2008

ABSTRACT

Cry proteins are a large family of crystalline toxins produced by
Bacillus thuringiensis
. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create “Crybodies.” Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins—a major class of toxin receptors—since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.
The Nascent Parasitophorous Vacuole Membrane of
Encephalitozoon cuniculi
Is Formed by Host Cell Lipids and Contains Pores Which Allow Nutrient Uptake
Authors: Rönnebäumer Karin , Gross Uwe , Bohne Wolfgang 
Published: April 2008

ABSTRACT

Microsporidia are obligate intracellular pathogens which enter host cells by the discharge of a hollow tube through which the sporoplasma is extruded into the host cell. Since this invasion mechanism is very different from common entry strategies, the formation of the parasitophorous vacuole (PV) in
Encephalitozoon
species is likely to be distinct from known principles. We investigated the origin of the nascent
Encephalitozoon cuniculi
PV membrane with the aid of fluorescent lipid probes. When Bodipy 500/510-C
12
-HPC-labeled spores were used for infection, the emerging PV membrane was unlabeled, suggesting that sporoplasma-derived lipids do not significantly contribute to the formation of the PV membrane. In contrast, when raft and nonraft microdomains of the host cell plasma membrane were selectively labeled with DiIC
16
and Speedy DiO, both tracers were detectable in the nascent PV membrane shortly after infection, indicating that the bulk lipids of the PV membrane are host cell derived. Time-lapse fluorescence microscopy revealed that the formation of the PV membrane is a fast event (<1.3 s), which occurred simultaneously with the extrusion of the sporoplasma. The portion of the discharged tube which is in contact with the host cell was found to be coated with labeled host cell lipids, which might be an indication for a plasma membrane invagination at the contact site. To investigate the presence of pores in the
E. cuniculi
PV membrane, we microinjected fluorescent dyes of different sizes into infected host cells. A 0.5-kDa dextran as well as 0.8- to 1.1-kDa peptides could rapidly enter the PV, while a 10-kDa dextran was stably excluded from the PV lumen, indicating that the PV membrane possesses pores with an exclusion size of <10 kDa, which should allow metabolite exchange.
Rtr1 Is the
Saccharomyces cerevisiae
Homolog of a Novel Family of RNA Polymerase II-Binding Proteins
Authors: Gibney Patrick A. , Fries Thomas , Bailer Susanne M. , Morano Kevin A. 
Published: April 2008

ABSTRACT

Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in
Saccharomyces cerevisiae
, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37°C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C
RTR1
(
r
egulator of
tr
anscription). The core RNAPII subunits
RPB5, RPB7
, and
RPB9
were isolated as potent high-copy-number suppressors of the
rtr1
Δ temperature-sensitive growth phenotype, and deletion of the nonessential subunits
RPB4
and
RPB9
hypersensitized cells to
RTR1
overexpression. Disruption of
RTR1
resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently,
rtr1
Δ cells are defective in inducible transcription from the
GAL1
promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.
A Novel Actin-Related Protein Is Associated with Daughter Cell Formation in
Toxoplasma gondii
Authors: Gordon Jennifer L. , Beatty Wandy L. , Sibley L. David 
Published: April 2008

ABSTRACT

Cell division in
Toxoplasma gondii
occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using
Toxoplasma
as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.
Transcriptional Repression by the Pho4 Transcription Factor Controls the Timing of
SNZ1
Expression
Authors: Nishizawa Masafumi , Komai Tae , Morohashi Nobuyuki , Shimizu Mitsuhiro , Toh-e Akio 
Published: April 2008

ABSTRACT

Nutrient-sensing kinases play important roles for the yeast
Saccharomyces cerevisiae
to adapt to new nutrient conditions when the nutrient status changes. Our previous global gene expression analysis revealed that the Pho85 kinase, one of the yeast nutrient-sensing kinases, is involved in the changes in gene expression profiles when yeast cells undergo a diauxic shift. We also found that the stationary phase-specific genes
SNZ1
and
SNO1
, whch share a common promoter, are not properly induced when Pho85 is absent. To examine the role of the kinase in
SNZ1
/
SNO1
regulation, we analyzed their expression during the growth of various yeast mutants, including those affecting Pho85 function or lacking the Pho4 transcription factor, an in vivo substrate of Pho85, and tested Pho4 binding by chromatin immunoprecipitation. Pho4 exhibits temporal binding to the
SNZ1
/
SNO1
promoter to down-regulate the promoter activity, and a Δ
pho4
mutation advances the timing of
SNZ1
/
SNO1
expression.
SNZ2
, another member of the
SNZ
/
SNO
family, is expressed at an earlier growth stage than
SNZ1
, and Pho4 does not affect this timing, although Pho85 is required for
SNZ2
expression. Thus, Pho4 appears to regulate the different timing of the expression of the
SNZ/SNO
family members. Pho4 binding to the
SNZ1
/
SNO1
promoter is accompanied by alterations in chromatin structure, and Rpd3 histone deacetylase is required for the proper timing of
SNZ1
/
SNO1
expression, while Asf1 histone chaperone is indispensable for their expression. These results imply that Pho4 plays positive and negative roles in transcriptional regulation, with both cases involving structural changes in its target chromatin.
MPL1, a Novel Phosphatase with Leucine-Rich Repeats, Is Essential for Proper ERK2 Phosphorylation and Cell Motility
Authors: Rodriguez Marbelys , Kim Bohye , Lee Nam-Sihk , Veeranki Sudhakar , Kim Leung 
Published: April 2008

ABSTRACTThe novelDictyosteliumphosphataseMPL1contains six leucine-rich repeats at the amino-terminal end and a phosphatase domain at the carboxyl end. Similarly architectured phosphatases exist among other protozoa, such asEntamoeba histolytica,Leishmania major, andTrypanosoma cruzi.MPL1was strongly induced after 5 h of development; ablation by homologous recombination led to defective streaming and aggregation during development. In addition, cyclic AMP (cAMP)-pulsedmpl1−cells showed reduced random and directional motility. At the molecular level,mpl1−cells displayed higher prestimulus and persistent poststimulus ERK2 phosphorylation in response to cAMP stimulation. Consistent with their phenotype of persistent ERK2 phosphorylation,mpl1−cells also displayed an aberrant pattern of cAMP production, resembling that of theregA−cells. Reintroduction of a full-lengthMPL1intompl1−cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild-type cells. We propose that MPL1 is a novel phosphatase essential for proper regulation of ERK2 phosphorylation and optimal motility during development.
Investigating Early Events in Receptor Binding and Translocation of Colicin E9 Using Synchronized Cell Killing and Proteolytic Cleavage
Authors: Zhang Ying , Vankemmelbeke Mireille N. , Holland Lisa E. , Walker David C. , James Richard , Penfold Christopher N. 
Published: April 2008

ABSTRACT

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of
Escherichia coli
and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9
s-s
) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9
s-s
, we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the
E. coli
cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9
AF
) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.
Transcriptional Regulation of the Nitrile Hydratase Gene Cluster in
Pseudomonas chlororaphis
B23
Authors: Sakashita Toshihide , Hashimoto Yoshiteru , Oinuma Ken-Ichi , Kobayashi Michihiko 
Published: April 2008

ABSTRACT

An enormous amount of nitrile hydratase (NHase) is inducibly produced by
Pseudomonas chlororaphis
B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the
P. chlororaphis
B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated. Recently, we reported that the NHase gene cluster comprises seven genes (
oxdA
,
amiA
,
nhpA
,
nhpB
,
nhpC
,
nhpS
, and
acsA
). Sequence analysis of the 1.5-kb region upstream of the
oxdA
gene revealed the presence of a 936-bp open reading frame (designated
nhpR
), which should encode a protein with a molecular mass of 35,098. The deduced amino acid sequence of the
nhpR
product showed similarity to the sequences of transcriptional regulators belonging to the XylS/AraC family. Although the transcription of the eight genes (
nhpR
,
oxdA
,
amiA
,
nhpABC
,
nhpS
, and
acsA
) in the NHase gene cluster was induced significantly in the
P. chlororaphis
B23 wild-type strain after addition of methacrylamide to the medium, transcription of these genes in the
nhpR
disruptant was not induced, demonstrating that
nhpR
codes for a positive transcriptional regulator in the NHase gene cluster. A reverse transcription-PCR experiment revealed that five genes (
oxdA
,
amiA
,
nhpA
,
nhpB
, and
nhpC
) are cotranscribed, as are two other genes (
nhpS
and
acsA
). The transcription start sites for
nhpR
,
oxdA
,
nhpA
, and
nhpS
were mapped by primer extension analysis, and putative −12 and −24 σ
54
-type promoter binding sites were identified. NhpR was found to be the first transcriptional regulator of NHase belonging to the XylS/AraC family.
Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-
co
-3-Hydroxyvalerate) Synthase in
Haloferax mediterranei
Authors: Lu Qiuhe , Han Jing , Zhou Ligang , Zhou Jian , Xiang Hua 
Published: April 2008

ABSTRACT

The haloarchaeon
Haloferax mediterranei
has shown promise for the economical production of poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain
Archaea
. In this study, we cloned the gene cluster (
phaEC
Hme
) encoding a polyhydroxyalkanoate (PHA) synthase in
H. mediterranei
CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the
phaE
Hme
and
phaC
Hme
genes were constitutively expressed, and both the PhaE
Hme
and PhaC
Hme
proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the
phaEC
Hme
genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the
phaEC
Hme
genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaC
Hme
was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaE
Hme
/PhaC
Hme
(1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaC
Hme
and PhaE
Hme
, accounted for the PHBV synthesis in
H. mediterranei
.
The
Aeromonas hydrophila wb
*
O34
Gene Cluster: Genetics and Temperature Regulation
Authors: Jimenez Natalia , Canals Rocío , Saló María Teresa , Vilches Silvia , Merino Susana , Tomás Juan M. 
Published: April 2008

ABSTRACT

The
Aeromonas hydrophila wb
*
O34
gene cluster of strain AH-3 (serotype O34) was cloned and sequenced. This cluster contains genes necessary for the production of O34-antigen lipopolysaccharide (LPS) in
A. hydrophila
. We determined, using either mutation or sequence homology, roles for the majority of genes in the cluster by using the chemical O34-antigen LPS structure obtained for strain AH-3. The O34-antigen LPS export system has been shown to be a Wzy-dependent pathway typical of heteropolysaccharide pathways. Furthermore, the production of
A. hydrophila
O34-antigen LPS in
Escherichia coli
K-12 strains is dependent on incorporation of the Gne enzyme (UDP-
N
-acetylgalactosamine 4-epimerase) necessary for the formation of UDP-galactosamine in these strains. By using rapid amplification of cDNA ends we were able to identify a transcription start site upstream of the terminal
wzz
gene, which showed differential transcription depending on the growth temperature of the strain. The Wzz protein is able to regulate the O34-antigen LPS chain length. The differential expression of this protein at different temperatures, which was substantially greater at 20°C than at 37°C, explains the previously observed differential production of O34-antigen LPS and its correlation with the virulence of
A. hydrophila
serotype O34 strains.
Unusual, Virulence Plasmid-Dependent Growth Behavior of
Yersinia enterocolitica
in Three-Dimensional Collagen Gels
Authors: Freund Sandra , Czech Beate , Trülzsch Konrad , Ackermann Nikolaus , Heesemann Jürgen 
Published: April 2008

ABSTRACT

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen
Yersinia enterocolitica
in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless
Y. enterocolitica
was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37°C. Motility at 37°C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located
Yersinia
adhesion gene
yadA
. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVΔ
yadA
-carrying yersiniae formed loosely packed microcolonies at 37°C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a
yopE-gfp
reporter, we demonstrated Ca
2+
-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.
Characterization of
abn2
(
yxiA
), Encoding a
Bacillus subtilis
GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation
Authors: Inácio José Manuel , de Sá-Nogueira Isabel 
Published: April 2008

ABSTRACT

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-α-1,5-
l
-arabinanase (EC 3.2.1.99) from
Bacillus subtilis
, encoded by the
yxiA
gene (herein renamed
abn2
) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in
B. subtilis
. Abn2 was overproduced in
Escherichia coli
, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-α-1,5-
l
-arabinan as the preferred substrate, the enzyme exhibited an apparent

K
m

of 2.0 mg ml
−1
and
V
max
of 0.25 mmol min
−1
mg
−1
at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of
abn2
. Two potential transcriptional start sites were identified by primer extension analysis, and both a σ
A
-dependent and a σ
H
-dependent promoter were located. Transcriptional fusion studies revealed that the expression of
abn2
is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally,
trans
-acting factors and
cis
elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of
B. subtilis
for optimal degradation of arabinopolysaccharides.