bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Characterization of the Folate Catabolic Enzyme
p
-Aminobenzoyl-Glutamate Hydrolase from
Escherichia coli
Authors: Green Jacalyn M. , Hollandsworth Ryan , Pitstick Lenore , Carter Eric L. 
Published: February 2010

ABSTRACT

The
abg
locus of the
Escherichia coli
chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product,
p
-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the
p
-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by
abgA
and
abgB
. One-step purification was accomplished using a plasmid carrying
abgAB
with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a

K
m

value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min
−1
mg
−1
. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the
E. coli
chromosome may encode a portion of a folate catabolism pathway.
Transcriptome Analysis of
Pseudomonas syringae
Identifies New Genes, Noncoding RNAs, and Antisense Activity
Authors: Filiatrault Melanie J. , Stodghill Paul V. , Bronstein Philip A. , Moll Simon , Lindeberg Magdalen , Grills George , Schweitzer Peter , Wang Wei , Schroth Gary P. , Luo Shujun , Khrebtukova Irina , Yang Yong , Thannhauser Theodore , Butcher Bronwyn G. , Cartinhour Samuel , Schneider David J. 
Published: February 2010

ABSTRACT
To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.
Characterization of Acp, a Peptidoglycan Hydrolase of
Clostridium perfringens
with
N
-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis
Authors: Camiade Emilie , Peltier Johann , Bourgeois Ingrid , Couture-Tosi Evelyne , Courtin Pascal , Antunes Ana , Chapot-Chartier Marie-Pierre , Dupuy Bruno , Pons Jean-Louis 
Published: February 2010

ABSTRACT

This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of
Clostridium perfringens
. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the
Bacillus subtilis
peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an
N
-acetylglucosaminidase activity. The study of
acp
expression showed a constant expression during growth, which suggested an important role of Acp in growth of
C. perfringens
. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of
C. perfringens
. A knockout
acp
mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the
acp
mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and
acp
mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall
N
-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.
Utilization of Lactose and Galactose by
Streptococcus mutans
: Transport, Toxicity, and Carbon Catabolite Repression
Authors: Zeng Lin , Das Satarupa , Burne Robert A. 
Published: February 2010

ABSTRACT

Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by
Streptococcus mutans
, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (
lac
) and Leloir (
gal
) pathways was performed in strain UA159. Deletion of each gene in the
lac
operon caused various alterations in expression of a

P
lacA

-cat
promoter fusion and defects in growth on either lactose (
lacA
,
lacB
,
lacF
,
lacE
, and
lacG
), galactose (
lacA
,
lacB
,
lacD
, and
lacG
) or both sugars (
lacA
,
lacB
, and
lacG
). Failure to grow in the presence of galactose or lactose by certain
lac
mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EII
Man
and EII
Lac
, respectively, were shown to be the only effective transporters of galactose in
S. mutans
. Furthermore, disruption of
manL
, encoding EIIAB
Man
, led to increased resistance to glucose-mediated CCR when lactose was used to induce the
lac
operon, but resulted in reduced
lac
gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.
Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium
Prevotella bryantii
B
1
4
Authors: Dodd Dylan , Kiyonari Shinichi , Mackie Roderick I. , Cann Isaac K. O. 
Published: February 2010

ABSTRACT

Prevotella bryantii
B
1
4 is a member of the phylum
Bacteroidetes
and contributes to the degradation of hemicellulose in the rumen. The genome of
P. bryantii
harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in
Escherichia coli
. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either
para
-nitrophenyl (
p
NP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the
k
cat
/

K
m

for
p
NP-β-
d
-xylopyranoside (
p
NPX) with a concurrent 1.1-fold increase in the
k
cat
/

K
m

for
p
NP-β-
d
-glucopyranoside (
p
NPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.
Back to the Future: Studying Cholera Pathogenesis Using Infant Rabbits
Authors: Ritchie Jennifer M. , Rui Haopeng , Bronson Roderick T. , Waldor Matthew K. 
Published: March 2010

ABSTRACT

Cholera is a severe diarrheal disease, caused by
Vibrio cholerae
, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of
V. cholerae
into 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the “rice-water stool” produced by cholera patients. As in humans,
V. cholerae
mutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-type
V. cholerae
was predominantly found in close association with mucin. Large aggregates of mucin-embedded
V. cholerae
were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences
V. cholerae
’s association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may govern
V. cholerae
infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic
V. cholerae
suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-
V. cholerae
vaccines.

IMPORTANCE
Cholera remains a significant threat to populations in developing nations. Currently, there is no reproducible, nonsurgical animal model of cholera, the secretory diarrheal disease caused by
Vibrio cholerae
. We found that oral infection of infant rabbits with
V. cholerae
led to lethal, watery diarrhea in most rabbits. Using this disease model, we discovered a new role for cholera toxin (CT) during infection. This toxin not only caused secretory diarrhea but also profoundly influenced how
V. cholerae
associates with the intestine and how the pathogen exits from the host. Rabbits inoculated with
V. cholerae
that does not produce CT developed mild diarrhea, suggesting that this model may prove useful for generating improved live attenuated-
V. cholerae
vaccine candidates. Overall, our findings suggest that the infant rabbit model will enable pursuit of several new avenues for research on cholera pathogenesis, as well as serve as a vehicle for testing new therapeutics.
Re–Os molybdenite dates from the Ballachulish and Kilmelford Igneous Complexes (Scottish Highlands): age constraints for late Caledonian magmatism
Authors: Conliffe J. , Selby D. , Porter S.J. , Feely M. 
Published: March 2010

Abstract:

New Re–Os molybdenite geochronology is presented from the Ballachulish Igneous Complex (433.5 ± 1.8 Ma) and the Kilmelford Igneous Complex (425.8 ± 1.7 Ma) of the SW Scottish Highlands. The sulphide mineralization in the Ballachulish Igneous Complex is related to the latest phase of granite magmatism, and therefore the new Re–Os age provides a minimum crystallization age,
c
. 3.7 Ma earlier than previous crystallization age estimates. This Re–Os age overlaps U–Pb ages obtained from subduction-related granitic and appinitic magmatism north of the Great Glen Fault, and an origin related to active subduction rather than slab breakoff is proposed for the Ballachulish Igneous Complex. Molybdenite mineralization in the Kilmelford Igneous Complex is spatially and genetically associated with porphyry Cu mineralization, which is consistent with the rapid ascent of volatile-rich magma during early rebound following the breakoff of subducted oceanic lithosphere. The Kilmelford Igneous Complex and the coeval Lorn Lava Pile may represent the earliest of the igneous bodies predicted by slab breakoff, indicating that slab breakoff occurred at
c
. 426 Ma.
The middle Darriwilian (Ordovician) δ
13
C excursion (MDICE) discovered in the Yangtze Platform succession in China: implications of its first recorded occurrences outside Baltoscandia
Authors: Schmitz Birger , Bergström Stig M. , Xiaofeng Wang 
Published: March 2010

Abstract:

The middle Darriwilian δ
13
C excursion (MDICE), one of the least known of the Ordovician δ
13
C excursions, has previously been recorded only from the Middle Ordovician of Baltoscandia. Analysis of many recently collected limestone samples from the Guniutan Formation at two Yangtze Platform localities show elevated δ
13
C values in the same biostratigraphic interval (
Microzarkodina ozarkodella
Conodont Subzone) as the MDICE in Baltoscandia, which justifies identification of the Chinese δ
13
C excursion as the MDICE. These occurrences, which are in strata that show striking lithological and conodont faunal similarity to the Swedish Holen Limestone and some coeval units in Estonia, indicate that the MDICE, the stratigraphically oldest of the named Ordovician δ
13
C excursions, is likely to have a world-wide distribution and to have great potential for local and long-range chemostratigraphic correlations.
Making Law for the Spirits: Angakkuit, Revelation and Rulemaking in the Canadian Arctic
Authors: Stone Thomas 
Published: February 2010

AbstractProcesses of revelation and rulemaking are examined in the context of the indigenous religion of the Inuit of arctic Canada. Instances of misfortune, conventionally understood to be the manner in which spirit intentions concerning human conduct are revealed, instigate social mechanisms through which normative rules are created and maintained. The performances of Inuit religious specialists, the Angakkuit, play a key role in this process. The Inuit case invites comparative assessments of means of rule construction and the discernment of intentions in the context of both other religious traditions and secular normative systems.
Beyond the Horizons of Legends:Traditional Imagery and Direct Experience in Medieval Accounts of Asia
Authors: Valtrová Jana 
Published: February 2010

AbstractThe article deals with several medieval travel accounts about Asia, which were produced during the 13th and 14th centuries, in the time of the so called Mongol mission. These reports were written by Franciscan and also some Dominican missionaries, namely William of Rubruck, John Plano of Carpini, Odoric of Pordenone, John of Marignola, Jordanus Catalanus and a few others. The aim of the article is to analyze the encounter of European travelers’ “traditional” ideas about Asia with the actual reality. Did the friars mostly rely on their anticipations, or were they open to new information, even if this could destroy views often advocated by eminent authorities of European medieval thought? The article analyses three “traditional” topoi, each of them in the context of the above-mentioned reports: earthly paradise, the kingdom of Prester John and human monsters. All of them belonged to the medieval lore regarding the East, as testified by many literary as well as pictorial documents. Each of the authors adopted a slightly different strategy for how to solve the potential conflicts between “tradition” and experience. Finally, I suggest conceptualizing the problem of “tradition” and experience in medieval travel accounts with reference to a typology of “otherness” created by Karlheinz Ohle. According to Ohle, a “cognitive Other” (1) is an unknown, never encountered Other which can only be imagined, whereas a “normative Other” (2), is an Other which is directly encountered and gradually explored. In my opinion, the friars’ medieval travel accounts actually reflect a shift from imagination towards gradual encounter and exploration — in these reports the imagined (cognitive) fabulous East gradually turned into an explored (normative) reality.
Participation and Giving Ultimate Meaning: Exploring the Entanglement of Psychology of Religion and Phenomenology of Religion in the Netherlands
Authors: Westerink Herman 
Published: February 2010

AbstractPsychology of religion in the Netherlands is rediscovering its historic entanglement with phenomenology of religion in the context of a current transition emancipating itself from the theological objective of re-establishing the relation between theology and faith practice (from the 1960s onwards), and developing into a discipline focusing on “lived religion” and interculturality in closer cooperation with religious studies. In this article the entanglement of psychology of religion and phenomenology of religion is explored starting with the writings of Gerardus van der Leeuw, his interest in a psychological method in phenomenology and his reception of Lévy-Bruhl’s concept of mystic participation. It is argued that psychology of religion in the Netherlands after the Second World War emerged out of the critique by Fokke Sierksma of the phenomenological method in the context of emancipating the science of religion (godsdienstwetenschap) from theology, and the reaction this provoked in the work of Han Fortmann, who defended Lévy-Bruhl and Van der Leeuw in order to “save” religiosity in a modern secularized world. This theological objective further colored developments in psychology of religion, notably the current discussion on “giving ultimate meaning” (zingeving). In the light of an expected closer cooperation between psychology of religion and religious studies, a critical reflection on the often unreflected theological positions and objectives in discussions on “giving ultimate meaning” is pleaded for.
The Human Rights of Minority Women: Romani Women's Rights from a Perspective on International Human Rights Law and Politics
Authors: Ravnbøl Camilla Ida 
Published: February 2010

AbstractThis article explores the complexities surrounding the human rights of minority women. With analytical focus on Romani women in Europe it seeks to contribute with new insight into the grey areas of rights issues, where groups within special rights categories share different human rights concerns, by being both women and members of a minority group. Through an investigation of how contemporary human rights law and politics serve to address the concerns of Romani women, it sheds light on the challenges that the Romani women's issue presents to the international human rights framework. These challenges go beyond the Romani issue only and into larger issues of women and minorities. It raises questions as to whether the historical separation between categories of gender and race/ethnicity within the international community in practice has become a gap that isolates Romani women from the human rights attention that they claim. It is argued that in order to strengthen the validity of human rights in the lives of Romani women, as a framework that ensures their full and equal protection, special attention needs to be given to interrelated grounds and forms of discrimination. “Intersectionality” is re-introduced as a concept to frame such new approaches to the human rights of Romani women. The article is a summary version of the thesis “The Human Rights of Minority Women: Challenging International Discourses with the Case of Romani Women”, for which the author was awarded the Martin Alexanderson Research Scholarship, administered by the Raoul Wallenberg Institute of Human Rights and Humanitarian Law in Lund, Sweden. This summary version brings forward the main arguments of the thesis which was an awarded EMA thesis 2006–2007 of the European University Institute in Venice. For this reason it does not present any new findings or data after 2007 but merely summarises the main chapters of the thesis. The thesis investigated the complexities surrounding Romani women's human rights at UN and European level. Thus, national systems and the regional systems in the Americas and Africa are excluded. The empirical data comes primarily from the European region.
Defining 'Indigenous' in Bangladesh: International Law in Domestic Context
Authors: Ahmed Kawser 
Published: February 2010

AbstractBangladesh is one of the 11 states which abstained in voting on the United Nations (UN) Declaration on the Rights of Indigenous Peoples. The reason as stated by the representative of Bangladesh at UN is that the term 'indigenous peoples' has not been clearly defined or identified in the aforementioned Declaration. In fact, the government of Bangladesh has been persistently denying many of the marginal communities' claim to recognition as indigenous peoples. The article argues that the state of non-dominance is one of the determining criteria of the definitions of indigenous peoples in international law. Drawing on the discourses of subaltern historiography and internal colonialism, this article further argues that the said marginal communities of Bangladesh indeed meet all the criteria including non-dominance inasmuch as they are entitled to recognition and legal protection as indigenous peoples. Case studies on historical profiles of three marginal communities of Bangladesh are provided as factual evidence in support of the above proposition.