bluuebunny/crossref_metadata_2025_split huggingface dataset:
Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of
Entamoeba histolytica
Authors: Panigrahi Sunil K. , Jhingan Gagan Deep , Som Indrani , Bhattacharya Alok , Petri William A. , Bhattacharya Sudha 
Published: November 2008

ABSTRACT

rRNA genes of
Entamoeba histolytica
are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions −86 to +123 (rDNA I) and positions −101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions −53 to −68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions.
Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the
Escherichia coli
Cell Division Machinery
Authors: Karimova Gouzel , Robichon Carine , Ladant Daniel 
Published: November 2008

ABSTRACT

Formation of the
Escherichia coli
division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the
E. coli
cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of
E. coli
cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many
E. coli
cell division proteins and to localize to the
E. coli
division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the
ftsQ1
(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the
E. coli
cell division machinery.
Transport of Phage P22 DNA across the Cytoplasmic Membrane
Authors: Perez Gerardo L. , Huynh Bao , Slater Miranda , Maloy Stanley 
Published: November 2008

ABSTRACT

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into
Salmonella
is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of
32
P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of
Salmonella
.
Heat-Labile Enterotoxin PromotesEscherichia coliAdherence to Intestinal Epithelial Cells
Authors: Johnson Amber M. , Kaushik Radhey S. , Francis David H. , Fleckenstein James M. , Hardwidge Philip R. 
Published: November 2008

ABSTRACTGiven recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenicEscherichia coli(ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase inE. coliadherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3′,5′-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.
Streptococcus mutans
SMU.623c Codes for a Functional, Metal-Dependent Polysaccharide Deacetylase That Modulates Interactions with Salivary Agglutinin
Authors: Deng Dong Mei , Urch Jonathan E. , ten Cate Jacob M. , Rao Vincenzo A. , van Aalten Daan M. F. , Crielaard Wim 
Published: November 2008

ABSTRACT

The genome sequence of the oral pathogen
Streptococcus mutans
predicts the presence of two putative polysaccharide deacetylases. The first, designated PgdA in this paper, shows homology to the catalytic domains of peptidoglycan deacetylases from
Streptococcus pneumoniae
and
Listeria monocytogenes
, which are both thought to be involved in the bacterial defense mechanism against human mucosal lysozyme and are part of the CAZY family 4 carbohydrate esterases.
S. mutans
cells in which the
pgdA
gene was deleted displayed a different colony texture and a slightly increased cell surface hydrophobicity and yet did not become hypersensitive to lysozyme as shown previously for
S. pneumoniae
. To understand this apparent lack of activity, the high-resolution X-ray structure of
S. mutans
PgdA was determined; it showed the typical carbohydrate esterase 4 fold, with metal bound in a His-His-Asp triad. Analysis of the protein surface showed that an extended groove lined with aromatic residues is orientated toward the active-site residues. The protein exhibited metal-dependent de-
N
-acetylase activity toward a hexamer of
N
-acetylglucosamine. No activity was observed toward shorter chitooligosaccharides or a synthetic peptidoglycan tetrasaccharide. In agreement with the lysozyme data this would suggest that
S. mutans
PgdA does not act on peptidoglycan but on an as-yet-unidentified polysaccharide within the bacterial cell surface. Strikingly, the
pgdA
-knockout strain showed a significant increase in aggregation/agglutination by salivary agglutinin, in agreement with this gene acting as a deacetylase of a cell surface glycan.
Erwinia chrysanthemiIron Metabolism: the Unexpected Implication of the Inner Membrane Platform within the Type II Secretion System
Authors: Douet Vanessa , Expert Dominique , Barras Frédéric , Py Béatrice 
Published: November 2008

ABSTRACTThe type II secretion (T2S) system is an essential device forErwinia chrysanthemivirulence. Previously, we reported the key role of the OutF protein in forming, along with OutELM, an inner membrane platform in the Out T2S system. Here, we report that OutF copurified with five proteins identified by matrix-assisted laser desorption ionization-time of flight analysis as AcsD, TogA, SecA, Tsp, and DegP. The AcsD protein was known to be involved in the biosynthesis of achromobactin, which is a siderophore important forE. chrysanthemivirulence. The yeast two-hybrid system allowed us to gain further evidence for the OutF-AcsD interaction. Moreover, we showed that lack of OutF produced a pleiotropic phenotype: (i) altered production of the two siderophores ofE. chrysanthemi, achromobactin and chrysobactin; (ii) hypersensitivity to streptonigrin, an iron-activated antibiotic; (iii) increased sensitivity to oxidative stress; and (iv) absence of the FbpA-like iron-binding protein in the periplasmic fraction. Interestingly,outEandoutLmutants also exhibited similar phenotypes, but,outDandoutJmutants did not. Moreover, using the yeast two-hybrid system, several interactions were shown to occur between components of the T2S system inner membrane platform (OutEFL) and proteins involved in achromobactin production (AcsABCDE). The OutL-AcsD interaction was also demonstrated by Ni2+affinity chromatography. These results fully confirm our previous view that the T2S machinery is made up of three discrete blocks. The OutEFLM-forming platform is proposed to be instrumental in two different processes essential for virulence, protein secretion and iron homeostasis.
Genetic Interactions of
smc
,
ftsK
, and
parB
Genes in
Streptomyces coelicolor
and Their Developmental Genome Segregation Phenotypes
Authors: Dedrick Rebekah M. , Wildschutte Hans , McCormick Joseph R. 
Published: November 2008

ABSTRACT

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for
Streptomyces coelicolor
, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for
ftsK
,
smc
, and
parB
. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (
s
tructural
m
aintenance of
c
hromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an
ftsK
-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the
smc
and
ftsK
deletion-insertion mutants (
ftsK
′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described
parB
-null mutant (12% anucleate spores).
parB
and
smc
were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the
smc parB
mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that
scpA
and
scpAB
mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.
AglC and AglK Are Involved in Biosynthesis and Attachment of Diacetylated Glucuronic Acid to the N-Glycan in
Methanococcus voltae
Authors: Chaban Bonnie , Logan Susan M. , Kelly John F. , Jarrell Ken F. 
Published: November 2008

ABSTRACT

Recent advances in the field of prokaryotic N-glycosylation have established a foundation for the pathways and proteins involved in this important posttranslational protein modification process. To continue the study of the
Methanococcus voltae
N-glycosylation pathway, characteristics of known eukaryotic, bacterial, and archaeal proteins involved in the N-glycosylation process were examined and used to select candidate
M. voltae
genes for investigation as potential glycosyl transferase and flippase components. The targeted genes were knocked out via linear gene replacement, and the resulting effects on N-glycan assembly were identified through flagellin and surface (S) layer protein glycosylation defects. This study reports the finding that deletion of two putative
M. voltae
glycosyl transferase genes, designated
aglC
(for
a
rchaeal
gl
ycosylation) and
aglK
, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway in
M. voltae
and the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells.
RpoH
II
Activates Oxidative-Stress Defense Systems and Is Controlled by RpoE in the Singlet Oxygen-Dependent Response in
Rhodobacter sphaeroides
Authors: Nuss Aaron M. , Glaeser Jens , Klug Gabriele 
Published: November 2008

ABSTRACT

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen (
1
O
2
). Here we show that the alternative sigma factor RpoH
II
is required for the expression of important defense factors and that deletion of

rpoH
II

leads to increased sensitivity against exposure to
1
O
2
and methylglyoxal in
Rhodobacter sphaeroides
. The gene encoding RpoH
II
is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoH
II
-type sigma factor, which is widely distributed in the
Alphaproteobacteria
. RpoH
II
-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides,
1
O
2
scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH
II
in
R. sphaeroides
.
Medium-Dependent Phenotypes of
Streptomyces coelicolor
with Mutations in
ftsI
or
ftsW
Authors: Bennett Jennifer A. , Yarnall Jennifer , Cadwallader Adam B. , Kuennen Rebecca , Bidey Peter , Stadelmaier Beth , McCormick Joseph R. 
Published: November 2008

ABSTRACT

Streptomyces coelicolor
A3(2)
ftsI
- and
ftsW
-null mutants produced aerial hyphae with no evidence of septation when grown on a traditional osmotically enhanced medium. This phenotype was partially suppressed when cultures were grown on media prepared without sucrose. We infer that functional FtsZ rings can form in
ftsI-
and
ftsW
-null mutants under certain growth conditions.
A Genomic Island of an Extraintestinal Pathogenic
Escherichia coli
Strain Enables the Metabolism of Fructooligosaccharides, Which Improves Intestinal Colonization
Authors: Schouler Catherine , Taki Ahmed , Chouikha Iman , Moulin-Schouleur Maryvonne , Gilot Philippe 
Published: November 2008

ABSTRACT

Prebiotics such as fructooligosaccharides (FOS) are increasingly being used in some countries for improving human and animal health and as an alternative to antibiotic growth promoters in animals, with various degrees of success. It has been observed that FOS stimulate the proliferation of probiotic bacteria and, at the same time, decrease the population of bacteria associated with disease. This observation assumes that pathogenic bacteria do not metabolize FOS and, therefore, lose their competitive advantage over beneficial bacteria. Here we present evidence that some pathogenic
Escherichia coli
strains can metabolize FOS and show that this property helps the bacterium colonize the intestine. These findings highlight the potential risk that a high level of prebiotic usage could lead to the emergence of well-adapted pathogenic strains that metabolize prebiotic substances.
Mutagenesis of Region 4 of Sigma 28 from
Chlamydia trachomatis
Defines Determinants for Protein-Protein and Protein-DNA Interactions
Authors: Hua Ziyu , Rao Xiancai , Feng Xiaogeng , Luo Xudong , Liang Yanmei , Shen Li 
Published: November 2008

ABSTRACT

Transcription factor σ
28
in
Chlamydia trachomatis
(σ
28
Ct
) plays a role in the regulation of genes that are important for late-stage morphological differentiation. In vitro mutational and genetic screening in
Salmonella enterica
serovar Typhimurium was performed in order to identify mutants with mutations in region 4 of σ
28
Ct
that were defective in σ
28
-specific transcription. Specially, the previously undefined but important interactions between σ
28
Ct
region 4 and the flap domain of the RNA polymerase β subunit (β-flap) or the −35 element of the chlamydial
hctB
promoter were examined. Our results indicate that amino acid residues E206, Y214, and E222 of σ
28
Ct
contribute to an interaction with the β-flap when σ
28
Ct
associates with the core RNA polymerase. These residues function in contacts with the β-flap similarly to their counterpart residues in
Escherichia coli
σ
70
. Conversely, residue Q236 of σ
28
Ct
directly binds the chlamydial
hctB
−35 element. The conserved counterpart residue in
E. coli
σ
70
has not been reported to interact with the −35 element of the σ
70
promoter. Observed functional disparity between σ
28
Ct
and σ
70
region 4 is consistent with their divergent properties in promoter recognition. This work provides new insight into understanding the molecular basis of gene regulation controlled by σ
28
Ct
in
C. trachomatis.
Type II Secretion System Secretin PulD Localizes in Clusters in theEscherichia coliOuter Membrane
Authors: Buddelmeijer Nienke , Krehenbrink Martin , Pecorari Frédéric , Pugsley Anthony P. 
Published: November 2008

ABSTRACTThe cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of theKlebsiella oxytocatype II secretion system outer membrane secretin PulD (PulD-mCherry) inEscherichia coliwas determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.
The Crp-Activated Small Noncoding Regulatory RNA CyaR (RyeE) Links Nutritional Status to Group Behavior
Authors: De Lay Nicholas , Gottesman Susan 
Published: November 2008

ABSTRACTSmall noncoding regulatory RNAs (sRNAs) play a key role in regulating the expression of many genes inEscherichia coliand other bacteria. Many of the sRNAs identified inE. colibind to mRNAs in an Hfq-dependent manner and stimulate or inhibit translation of the mRNAs. Several sRNAs are regulated by well-studied global regulators. Here, we report characterization of the CyaR (RyeE) sRNA, which was previously identified in a global search for sRNAs inE. coli. We demonstrated that CyaR is positively regulated by the global regulator Crp under conditions in which cyclic AMP levels are high. We showed by using microarray analysis and Northern blotting that several genes are negatively regulated by CyaR, includingompX, encoding a major outer membrane protein;luxS, encoding the autoinducer-2 synthase;nadE, encoding an essential NAD synthetase; andyqaE, encoding a predicted membrane protein with an unknown function. Using translationallacZfusions toyqaE,ompX,nadE, andluxS, we demonstrated that the negative regulation of these genes by CyaR occurs at the posttranscriptional level and is direct. Different portions of a highly conserved 3′ region of CyaR are predicted to pair with sequences near the ribosome binding site of each of these targets; mutations in this sequence affected regulation, and compensatory mutations in the target mRNA restored regulation, confirming that there is direct regulation by the sRNA. These results provide insight into the mechanisms by which Crp negatively regulates genes such asluxSandompXand provide a link between catabolite repression, quorum sensing, and nitrogen assimilation inE. coli.