bluuebunny/crossref_metadata_2025_split huggingface dataset:
CalA, a Cyanobacterial AbrB Protein, Interacts with the Upstream Region of
hypC
and Acts as a Repressor of Its Transcription in the Cyanobacterium
Nostoc
sp. Strain PCC 7120
Authors: Agervald Åsa , Zhang Xiaohui , Stensjö Karin , Devine Ellenor , Lindblad Peter 
Published: December 2009

ABSTRACT

The filamentous, heterocystous, nitrogen-fixing cyanobacterium
Nostoc
sp. strain PCC 7120 may contain, depending on growth conditions, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, CalA (Alr0946 in the genome), belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of
hypC
. Transcriptional investigations showed that
calA
is cotranscribed with the downstream gene
alr0947
, which encodes a putative protease from the abortive infection superfamily, Abi. CalA was shown to interact specifically not only with the upstream region of
hypC
but also with its own upstream region, acting as a repressor on
hypC
. The bidirectional hydrogenase activity was significantly downregulated when CalA was overexpressed, demonstrating a correlation with the transcription factor, either direct or indirect.
In silico
studies showed that homologues to both CalA and Alr0947 are highly conserved proteins within cyanobacteria with very similar physical organizations of the corresponding structural genes. Possible functions of the cotranscribed downstream protein Alr0947 are presented. In addition, we present a three-dimensional (3D) model of the DNA binding domain of CalA and putative DNA binding mechanisms are discussed.
A
m
ariner
-Based Transposon System for
In Vivo
Random Mutagenesis of
Clostridium difficile
Authors: Cartman Stephen T. , Minton Nigel P. 
Published: December 2009

ABSTRACT

Understanding the molecular basis of
Clostridium difficile
infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of
C. difficile
, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel
mariner
-based transposon system for
in vivo
random mutagenesis of
C. difficile
R20291, the BI/NAP1/027 epidemic strain at the center of the
C. difficile
outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (±0.4) × 10
−4
per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a
C. difficile
R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene
cspBA
and an auxotroph with an insertion in the pyrimidine biosynthesis gene
pyrB
. These results validate our
mariner
-based transposon system for use in forward genetic studies of
C. difficile
.
Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers
Authors: Castro Hector F. , Classen Aimée T. , Austin Emily E. , Norby Richard J. , Schadt Christopher W. 
Published: December 2009

ABSTRACT

Researchers agree that climate change factors such as rising atmospheric [CO
2
] and warming will likely interact to modify ecosystem properties and processes. However, the response of the microbial communities that regulate ecosystem processes is less predictable. We measured the direct and interactive effects of climatic change on soil fungal and bacterial communities (abundance and composition) in a multifactor climate change experiment that exposed a constructed old-field ecosystem to different atmospheric CO
2
concentration (ambient, +300 ppm), temperature (ambient, +3°C), and precipitation (wet and dry) might interact to alter soil bacterial and fungal abundance and community structure in an old-field ecosystem. We found that (i) fungal abundance increased in warmed treatments; (ii) bacterial abundance increased in warmed plots with elevated atmospheric [CO
2
] but decreased in warmed plots under ambient atmospheric [CO
2
]; (iii) the phylogenetic distribution of bacterial and fungal clones and their relative abundance varied among treatments, as indicated by changes in 16S rRNA and 28S rRNA genes; (iv) changes in precipitation altered the relative abundance of
Proteobacteria
and
Acidobacteria
, where
Acidobacteria
decreased with a concomitant increase in the
Proteobacteria
in wet relative to dry treatments; and (v) changes in precipitation altered fungal community composition, primarily through lineage specific changes within a recently discovered group known as soil clone group I. Taken together, our results indicate that climate change drivers and their interactions may cause changes in bacterial and fungal overall abundance; however, changes in precipitation tended to have a much greater effect on the community composition. These results illustrate the potential for complex community changes in terrestrial ecosystems under climate change scenarios that alter multiple factors simultaneously.
Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF
Authors: Schilde Christina , Schönemann Barbara , Sehring Ivonne M. , Plattner Helmut 
Published: December 2009

ABSTRACT

We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named
Paramecium tetraurelia
synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal.
NSF
silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.
tRNA Gene Sequences Are Required for Transcriptional Silencing in Entamoeba histolytica
Authors: Irmer Henriette , Hennings Ina , Bruchhaus Iris , Tannich Egbert 
Published: December 2009

ABSTRACT

Transcriptional silencing by
trans
inactivation can contribute to the regulation of gene expression in eukaryotic cells. In the human intestinal protozoan parasite
Entamoeba histolytica
,
trans
inactivation of the amoebapore-A gene (
AP-A
) was recently achieved by episomal transfection of
E. histolytica
trophozoites with the plasmid psAP1. The mechanism of
AP-A trans
inactivation is largely unknown, though it was suggested that a partial short interspersed transposable element (SINE) is required. By systematic assessment of various
E. histolytica
isolates transfected with psAP1 derivates,
trans
inactivation of
AP-A
was restricted to the strain HM-1:IMSS (2411) but could not be achieved in other standard laboratory strains. Importantly, sequences of an
E. histolytica
tRNA array that were located on psAP1 in close proximity to the
AP-A
upstream region and comprising the glutamic acid (TTC) (E) and tyrosine (GTA) (Y) tRNA genes were indispensable for
AP-A
silencing. In contrast to the case described in previous reports, SINE was not required for
AP-A trans
inactivation.
AP-A
expression could be regained in silenced cells by episomal transfection under the control of a heterologous
E. histolytica
promoter, opening a way toward future silencing of individual genes of interest in
E. histolytica
. Our results indicate that tRNA gene-mediated silencing is not restricted to
Saccharomyces cerevisiae
.
Comparative Transcript Profiling of Candida albicans and Candida dubliniensis Identifies
SFL2
, a C. albicans Gene Required for Virulence in a Reconstituted Epithelial Infection Model
Authors: Spiering Martin J. , Moran Gary P. , Chauvel Murielle , MacCallum Donna M. , Higgins Judy , Hokamp Karsten , Yeomans Tim , d'Enfert Christophe , Coleman David C. , Sullivan Derek J. 
Published: December 2009

ABSTRACT

Candida albicans
and
Candida dubliniensis
are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel
C. albicans
virulence genes.
C. albicans
gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE).
C. albicans
(SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage.
C. dubliniensis
(CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in
C. dubliniensis
were upregulated in
C. albicans
. One such gene,
SFL2
(
orf19.3969
), encoding a putative heat shock factor, was deleted in
C. albicans
. ΔΔ
sfl2
cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of
SFL2
into the ΔΔ
sfl2
strain. Moreover,
SFL2
overexpression in
C. albicans
triggered hyphal morphogenesis. Although
SFL2
deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔ
sfl2
strain-infected kidney tissues contained only yeast cells. These results suggest a role for
SFL2
in morphogenesis and an indirect role in
C. albicans
pathogenesis in epithelial tissues.
Hgc1 Mediates Dynamic Candida albicans-Endothelium Adhesion Events during Circulation
Authors: Wilson Duncan , Hube Bernhard 
Published: December 2009

ABSTRACT

Common iatrogenic procedures can result in translocation of the human pathogenic fungus
Candida albicans
from mucosal surfaces to the bloodstream. Subsequent disseminated candidiasis and infection of deep-seated organs may occur if the fungus is not eliminated by blood cells. In these cases, fungal cells adhere to the endothelial cells of blood vessels, penetrate through endothelial layers, and invade deeper tissue. In this scenario, endothelial adhesion events must occur during circulation under conditions of physiological blood pressure. To investigate the fungal and host factors which contribute to this essential step of disseminated candidiasis, we have developed an
in vitro
circulatory
C. albicans
-endothelium interaction model. We demonstrate that both
C. albicans
yeast and hyphae can adhere under flow at a pressure similar to capillary blood pressure. Serum factors significantly enhanced the adhesion potential of viable but not killed
C. albicans
cells to endothelial cells. During circulation,
C. albicans
cells produced hyphae and the adhesion potential first increased, then decreased with time. We provide evidence that a specific temporal event in the yeast-to-hyphal transition, regulated by the G
1
cyclin Hgc1, is critical for
C. albicans
-endothelium adhesion during circulation.
Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of
Entamoeba histolytica
Authors: Moreno Heriberto , Linford Alicia S. , Gilchrist Carol A. , Petri William A. 
Published: December 2009

ABSTRACT

The
Entamoeba histolytica
upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca
2+
-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca
2+
-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca
2+
. Recombinant and native EhC2A bound phospholipid liposomes in a Ca
2+
-dependent manner, with half-maximal binding occurring at 3.4 μM free Ca
2+
. A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca
2+
-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca
2+
concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca
2+
-dependent anchoring of the transcription factor to the amebic plasma membrane.
Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of
Campylobacter jejuni
Authors: Gaasbeek Esther J. , Wagenaar Jaap A. , Guilhabert Magalie R. , van Putten Jos P. M. , Parker Craig T. , van der Wal Fimme J. 
Published: December 2009

ABSTRACT

The species
Campylobacter jejuni
is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by
dns
, inhibits natural transformation in
C. jejuni
. In the present study, genetic factors coding for DNase activity in the absence of
dns
were identified. DNA arrays indicated that nonnaturally transformable
dns
-negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on
C. jejuni
integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative
Escherichia coli
and
C. jejuni
strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable
C. jejuni
strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the
C. jejuni
DNases belong to the
Serratia
nuclease family. Overall, the data indicate that the acquisition of prophage-encoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of
C. jejuni
through hydrolysis of DNA.
The MG1363 and IL1403 Laboratory Strains of
Lactococcus lactis
and Several Dairy Strains Are Diploid
Authors: Michelsen Ole , Hansen Flemming G. , Albrechtsen Bjarne , Jensen Peter Ruhdal 
Published: December 2009

ABSTRACT

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of
Lactococcus lactis
, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.
Genome-Wide Identification of Targets for the Archaeal Heat Shock Regulator Phr by Cell-Free Transcription of Genomic DNA
Authors: Keese Annette M. , Schut Gerrit J. , Ouhammouch Mohamed , Adams Michael W. W. , Thomm Michael 
Published: December 2009

ABSTRACT

The hyperthermophilic archaeon
Pyrococcus furiosus
grows optimally near 100°C and undergoes a heat shock response at 105°C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA
+
ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of
in vitro
RNA to
P. furiosus
whole-genome microarrays. Our results confirmed the
phr
, the
hsp20
, and the
aaa
+
ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5′-TTTAnnnACnnnnnGTnAnnAAAA-3′ (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.
The
Mycobacterium tuberculosis
DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy
Authors: Leistikow Rachel L. , Morton Russell A. , Bartek Iona L. , Frimpong Isaac , Wagner Karleen , Voskuil Martin I. 
Published: December 2009

ABSTRACT

Mycobacterium tuberculosis
survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The
M. tuberculosis
DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for
M. tuberculosis
survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once
M. tuberculosis
exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for
M. tuberculosis
to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.
Rhizobium leguminosarum hupE
Encodes a Nickel Transporter Required for Hydrogenase Activity
Authors: Brito Belén , Prieto Rosa-Isabel , Cabrera Ezequiel , Mandrand-Berthelot Marie-Andrée , Imperial Juan , Ruiz-Argüeso Tomás , Palacios José-Manuel 
Published: December 2009

ABSTRACT

Synthesis of the hydrogen uptake (Hup) system in
Rhizobium leguminosarum
bv.
viciae
requires the function of an 18-gene cluster (
hupSLCDEFGHIJK
-
hypABFCDEX
). Among them, the
hupE
gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of
hupE2
, a hydrogenase-unlinked gene identified in the
R. leguminosarum
genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the
Escherichia coli nikABCDE
mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX
5
DH and FHGX[AV]HGXE) that are required for HupE functionality. An
R. leguminosarum
double mutant, SPF22A (
hupE hupE2
), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (
Lens culinaris
L.) root nodules but not in pea (
Pisum sativum
L.) bacteroids. Moreover, heterologous expression of the
R. leguminosarum hup
system in bacteroid cells of
Rhizobium tropici
and
Mesorhizobium loti
displayed reduced levels of hydrogen uptake in the absence of
hupE
. These data support the role of
R. leguminosarum
HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.
Purification of
Helicobacter pylori
NCTC 11637 Cytochrome
bc
1
and Respiration with
d
-Proline as a Substrate
Authors: Tanigawa Minoru , Shinohara Tomomitsu , Nishimura Katsushi , Nagata Kumiko , Ishizuka Morio , Nagata Yoko 
Published: December 2009

ABSTRACT

Helicobacter pylori
is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found
d
-amino acid dehydrogenase-mediated electron transport from
d
-proline or
d
-alanine to oxygen via the respiratory chain in
H. pylori
. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the
d
-amino acids to oxygen using the recombinant cytochrome
bc
1
complex, cytochrome
c
-553, and the terminal oxidase cytochrome
cbb
3
complex. Upon addition of the recombinant
d
-amino acid dehydrogenase and
d
-proline or
d
-alanine to the reconstituted electron transport system, reduction of cytochrome
cbb
3
and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of
H. pylori
's electron transport chain, only the cytochrome
bc
1
complex had been remained unpurified. Therefore, we cloned and sequenced the
H. pylori
NCTC 11637 cytochrome
bc
1
gene clusters encoding Rieske Fe-S protein, cytochrome
b
, and cytochrome
c
1
, with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome
bc
1
complex showed an α peak at 561 nm with a shoulder at 552 nm.