bluuebunny/crossref_metadata_2025 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

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

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

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

Given 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

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

The 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

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

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

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

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

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

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

The 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

Small 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.
MtrR Modulates
rpoH
Expression and Levels of Antimicrobial Resistance in
Neisseria gonorrhoeae
Authors: Folster Jason P. , Johnson Paul J. T. , Jackson Lydgia , Dhulipali Vijaya , Dyer David W. , Shafer William M.
Published: November 2008

The MtrR transcriptional-regulatory protein is known to repress transcription of the
mtrCDE
operon, which encodes a multidrug efflux pump possessed by
Neisseria gonorrhoeae
that is important in the ability of gonococci to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to determine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated.
rpoH
, which encodes the general stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by MtrR, as it was found to bind to a DNA sequence upstream of
rpoH
that included sites within the
rpoH
promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely indirect and a reflection of MtrR control of
rpoH
expression. Inducible expression of MtrR was found to repress
rpoH
expression and to increase gonococcal susceptibility to hydrogen peroxide (H
2
O
2
) and an antibiotic (erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability to control the expression of the
mtrCDE
-encoded efflux pump operon and, as a consequence, levels of gonococcal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability of MtrR to regulate the expression levels of
rpoH
and RpoH-regulated genes also modulates levels of gonococcal susceptibility to H
2
O
2
.
Expression and Physiological Relevance of
Agrobacterium tumefaciens
Phosphatidylcholine Biosynthesis Genes
Authors: Klüsener Sonja , Aktas Meriyem , Thormann Kai M. , Wessel Mirja , Narberhaus Franz
Published: November 2008

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In
Rhizobiaceae
, including the phytopathogenic bacterium
Agrobacterium tumefaciens
, PC is essential for the establishment of a successful host-microbe interaction.
A. tumefaciens
produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes,
pmtA
(coding for a phospholipid
N
-methyltransferase) and
pcs
(coding for a PC synthase), are located on the circular chromosome of
A. tumefaciens
C58. Recombinant expression of
pmtA
and
pcs
in
Escherichia coli
revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in
A. tumefaciens
. The amount of PC in
A. tumefaciens
membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in
A. tumefaciens
.
A Genomic Distance Based on MUM Indicates Discontinuity between Most Bacterial Species and Genera
Authors: Deloger Marc , El Karoui Meriem , Petit Marie-Agnès
Published: November 2008

The fundamental unit of biological diversity is the species. However, a remarkable extent of intraspecies diversity in bacteria was discovered by genome sequencing, and it reveals the need to develop clear criteria to group strains within a species. Two main types of analyses used to quantify intraspecies variation at the genome level are the average nucleotide identity (ANI), which detects the DNA conservation of the core genome, and the DNA content, which calculates the proportion of DNA shared by two genomes. Both estimates are based on BLAST alignments for the definition of DNA sequences common to the genome pair. Interestingly, however, results using these methods on intraspecies pairs are not well correlated. This prompted us to develop a genomic-distance index taking into account both criteria of diversity, which are based on DNA maximal unique matches (MUM) shared by two genomes. The values, called MUMi, for MUM index, correlate better with the ANI than with the DNA content. Moreover, the MUMi groups strains in a way that is congruent with routinely used multilocus sequence-typing trees, as well as with ANI-based trees. We used the MUMi to determine the relatedness of all available genome pairs at the species and genus levels. Our analysis reveals a certain consistency in the current notion of bacterial species, in that the bulk of intraspecies and intragenus values are clearly separable. It also confirms that some species are much more diverse than most. As the MUMi is fast to calculate, it offers the possibility of measuring genome distances on the whole database of available genomes.
The
Anaplasma phagocytophilum
PleC Histidine Kinase and PleD Diguanylate Cyclase Two-Component System and Role of Cyclic Di-GMP in Host Cell Infection
Authors: Lai Tzung-Huei , Kumagai Yumi , Hyodo Mamoru , Hayakawa Yoshihiro , Rikihisa Yasuko
Published: November 2008

Anaplasma phagocytophilum
, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of
A. phagocytophilum
and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both
pleC
and
pleD
were expressed in an HGA patient. During
A. phagocytophilum
development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other
A. phagocytophilum
recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of
A. phagocytophilum
lysate with c-di-[
32
P]GMP detected an ∼47-kDa endogenous protein, presumably c-di-GMP downstream receptor. A new hydrophobic c-di-GMP derivative, 2′-
O
-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited
A. phagocytophilum
infection in HL-60 cells. Our results suggest that the two-component PleC-PleD system is a diguanylate cyclase and that a c-di-GMP-receptor complex regulates
A. phagocytophilum
intracellular infection.
The pH-Responsive Regulon of HP0244 (FlgS), the Cytoplasmic Histidine Kinase of
Helicobacter pylori
Authors: Wen Yi , Feng Jing , Scott David R. , Marcus Elizabeth A , Sachs George
Published: November 2008

Helicobacter pylori
colonizes the acidic gastric environment, in contrast to all other neutralophiles, whose acid resistance and tolerance responses allow only gastric transit. This acid adaptation is dependent on regulation of gene expression in response to pH changes in the periplasm and cytoplasm. The cytoplasmic histidine kinase, HP0244, which until now was thought only to regulate flagellar gene expression via its cognate response regulator, HP0703, was found to generate a response to declining medium pH. Although not required for survival at pH 4.5, HP0244 is required for survival at pH 2.5 with 10 mM urea after 30 min. Transcriptional profiling of a HP0244 deletion mutant grown at pH 7.4 confirmed the contribution of HP0244 to σ
54
activation via HP0703 to coordinate flagellar biosynthesis by a pH-independent regulon that includes 14 flagellar genes. Microarray analysis of cells grown at pH 4.5 without urea revealed an additional 22 genes, including 4 acid acclimation genes (
ureA
,
ureB
,
ureI
, and
amiE
) that are positively regulated by HP0244. Additionally, 86 differentially expressed genes, including 3 acid acclimation genes (
ureF
,
rocF
[arginase], and
ansB
[asparaginase]), were found in cells grown at pH 2.5 with 30 mM urea. Hence, HP0244 has, in addition to the pH-independent flagellar regulon, a pH-dependent regulon, which allows adaptation to a wider range of environmental acid conditions. An acid survival study using an HP0703 mutant and an electrophoretic mobility shift assay with in vitro-phosphorylated HP0703 showed that HP0703 does not contribute to acid survival and does not bind to the promoter regions of several genes in the HP0244 pH-dependent regulon, suggesting that there is a pathway outside the HP0703 regulon which transduces the acid-responsive signal sensed by HP0244.
DegU and Spo0A Jointly Control Transcription of Two Loci Required for Complex Colony Development byBacillus subtilis
Authors: Verhamme Daniel T. , Murray Ewan J. , Stanley-Wall Nicola R.
Published: November 2008

Biofilm formation is an example of a multicellular process which depends on cooperative behavior and differentiation within a bacterial population. Our findings indicate that there is a complex feedback loop that maintains the stoichiometry of the extracellular matrix and other proteins required for complex colony development byBacillus subtilis. Analysis of the transcriptional regulation of two DegU-activated genes that are required for complex colony development byB. subtilisrevealed additional involvement of global regulators that are central to controlling biofilm formation. Activation of transcription from both theyvcAandyuaBpromoters requires DegU∼phosphate, but transcription is inhibited by direct AbrB binding to the promoter regions. Inhibition of transcription by AbrB is relieved when Spo0A∼phosphate is generated due to its known role in inhibitingabrBexpression. Deletion of SinR, a key coordinator of motility and biofilm formation, enhanced transcription from both loci; however, no evidence of a direct interaction with SinR for either theyvcAoryuaBpromoter regions was observed. The enhanced transcription in thesinRmutant background was subsequently demonstrated to be dependent on biosynthesis of the polysaccharide component that forms the major constituent of theB. subtilisbiofilm matrix. Together, these findings indicate that a genetic network dependent on activation of both DegU and Spo0A controls complex colony development byB. subtilis.
Trends in Prokaryotic Evolution Revealed by Comparison of Closely Related Bacterial and Archaeal Genomes
Authors: Novichkov Pavel S. , Wolf Yuri I. , Dubchak Inna , Koonin Eugene V.
Published: November 2008

In order to explore microevolutionary trends in bacteria and archaea, we constructed a data set of 41 alignable tight genome clusters (ATGCs). We show that the ratio of the medians of nonsynonymous to synonymous substitution rates (dN/dS) that is used as a measure of the purifying selection pressure on protein sequences is a stable characteristic of the ATGCs. In agreement with previous findings, parasitic bacteria, notwithstanding the sometimes dramatic genome shrinkage caused by gene loss, are typically subjected to relatively weak purifying selection, presumably owing to relatively small effective population sizes and frequent bottlenecks. However, no evidence of genome streamlining caused by strong selective pressure was found in any of the ATGCs. On the contrary, a significant positive correlation between the genome size, as well as gene size, and selective pressure was observed, although a variety of free-living prokaryotes with very close selective pressures span nearly the entire range of genome sizes. In addition, we examined the connections between the sequence evolution rate and other genomic features. Although gene order changes much faster than protein sequences during the evolution of prokaryotes, a strong positive correlation was observed between the “rearrangement distance” and the amino acid distance, suggesting that at least some of the events leading to genome rearrangement are subjected to the same type of selective constraints as the evolution of amino acid sequences.
The LuxR Family Quorum-Sensing Activator MrtR Requires Its Cognate Autoinducer for Dimerization and Activation but Not for Protein Folding
Authors: Yang Menghua , Giel Jennifer L. , Cai Tao , Zhong Zengtao , Zhu Jun
Published: November 2008

MrtR, a LuxR homolog in
Mesorhizobium tianshanense
, is important for symbiosis. We found that MrtR requires its cognate
N
-acylhomoserine lactone for forming dimers, binding to a single DNA site and activating the downstream promoter. However, MrtR is able to fold independently of its ligand.
Relaxed Specificity of the
Bacillus subtilis
TatAdCd Translocase in Tat-Dependent Protein Secretion
Authors: Eijlander Robyn T. , Jongbloed Jan D. H. , Kuipers Oscar P.
Published: November 2008

Protein translocation via the twin arginine translocation (TAT) pathway is characterized by the translocation of prefolded proteins across the hydrophobic lipid bilayer of the membrane. In
Bacillus subtilis
, two different Tat translocases are involved in this process, and both display different substrate specificities: PhoD is secreted via TatAdCd, whereas YwbN is secreted via TatAyCy. It was previously assumed that both TatAy and TatCy are essential for the translocation of the YwbN precursor. Through complementation studies, we now show that TatAy can be functionally replaced by TatAd when the latter is offered to the cells in excess amounts. Moreover, under conditions of overproduction, TatAdCd, in contrast to TatAyCy, shows an increased tolerance toward the acceptance of various Tat-dependent proteins.
Maximal Efficiency of Coupling between ATP Hydrolysis and Translocation of Polypeptides Mediated by SecB Requires Two Protomers of SecA
Authors: Mao Chunfeng , Hardy Simon J. S. , Randall Linda L.
Published: November 2008

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in
Escherichia coli
during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.
A water-soluble carbon monoxide-releasing molecule (CORM-3) lowers intraocular pressure in rabbits
Authors: Stagni E , Privitera M G , Bucolo C , Leggio G M , Motterlini R , Drago F
Published: November 2008

Background:

Carbon monoxide-releasing molecules (CORMs) are a novel group of substances that are capable of modulating physiological functions via the liberation of CO.

Aims:

This study was undertaken to investigate the effects of CORM-3, a water-soluble CO-releasing agent, on two rabbit models of ocular hypertension.

Methods:

Ocular hypertension was induced by injecting α-chymotrypsin in the rabbit eye. The dose–response effect of CORM-3 on IOP was assessed by topical administration of the drug (0.001, 0.01, 0.1 and 1%). Ocular hypertension was also obtained by weekly subconjunctival injection of betamethasone, and animals were treated topically with CORM-3. A group of animals in both models was treated with the inactive form of the drug (iCORM-3).

Results:

CORM-3 induced a dose-dependent reduction in IOP in rabbits treated with α-chymotrypsin. A similar reduction in IOP was observed in rabbits with betamethasone-induced ocular hypertension treated with the drug. Treatment with the iCORM-3 had no effect on IOP in both models.

Conclusions:

Treatment with CORM-3 is associated with a reduction in IOP in two different rabbit models of ocular hypertension. These results support previous findings on the effect of haem oxygenase-derived CO on IOP and suggest a direct involvement of CO system in the regulation of ocular pressure probably through the modulation of aqueous humour dynamics.
Rapid detection and quantification of Propionibacteriaceae
Authors: Goldschmidt P , Ferreira C Costa , Degorge S , Benallaoua D , Boutboul S , Laroche L , Batellier L , Chaumeil C
Published: November 2008

Background:

Propionibacteriaceae (Propioni) are anaerobic bacteria associated with human and animal infections. Present-day methods of diagnosis for Propioni are unsatisfactory due to a lack of sensitivity of culture, time required for culture results (3 to 14 days) and difficulties in interpreting SYBR Green real-time PCR results. The goal of this work was to validate a new rapid and sensitive test for the diagnosis of Propioni infections (endophthalmitis, corneal ulcers and others).

Material and methods:

DNA was extracted using the MagNA Pure isolation kit (Roche), and bacterial detection and quantification were carried out with a set of original primers and probe (5′ATACGTAGGGTGCGAGCGTTGTCC; 5′TGGTGTTCCTCCTGATATCTGCGC and [Amino C6+JOE]-GATCGCGTCGGAAGTGTAATCTTGGGG-Black Hole Quencher). The PCR cycling programme consisted of one cycle at 95°C, 20 s and 45 cycles at 95°C, 3 s and 30 s at 60°C. DNA extraction yields were assessed in the same tube.

Results:

This test detects as few as 0.01 Equivalent PFU/μl Propioni in phosphate-buffered saline (PBS), aqueous humour, vitreous or cell suspensions. Propioni is detected as a single contaminant or mixed with other bacteria, fungi or human cells.

Conclusion:

The new real-time PCR is able to detect 0.01 Eq/CFU μl of Propioni suspended in PBS, vitreous, aqueous humour and human cells in less than 1.30 h.
Molecular pathology of endometrial carcinoma: practical aspects from the diagnostic and therapeutic viewpoints
Authors: Llobet D , Pallares J , Yeramian A , Santacana M , Eritja N , Velasco A , Dolcet X , Matias-Guiu X
Published: November 2008

This article reviews the main molecular alterations involved in endometrial carcinoma. Five molecular features (microsatellite instability, and mutations in the PTEN, k-RAS, PIK3CA and β-catenin genes) are characteristic of endometrioid carcinomas, whereas non-endometrioid carcinomas show alterations of p53, loss of heterozygosity (LOH) on several chromosomes, as well as other molecular alterations (STK15, p16, E-cadherin and C-erb B2). The review also covers the phenomenon of apoptosis resistance, as well as the results obtained from cDNA array studies, and the perspectives for targeted therapies. A group of practical applications of molecular pathology techniques are also mentioned: diagnosis of hereditary non-polyposis colon cancer syndrome in patients with endometrial carcinoma; evaluation of precursor lesions; prognosis; diagnosis, particularly for synchronous endometrioid carcinomas of the uterus and the ovaries; and targeted therapies.
New time-dependent approach to analyse the prognostic significance of immunohistochemical biomarkers in colon cancer and diffuse large B-cell lymphoma
Authors: Adams H , Tzankov A , Lugli A , Zlobec I
Published: November 2008

Aims:

Receiver operating characteristic (ROC) curve analysis is a well-established method to study the accuracies of biological markers. It may, however, be suboptimal for analysing outcomes over time, such as prognosis. Here, the clinical value of time-dependent ROC curve analysis for improving the identification of high-risk patients with colon cancers and diffuse large B-cell lymphomas (DLBCL) is explored.

Methods:

Using tissue microarrays, immunohistochemistry was performed on two matched sets (N = 469, each) of colon cancers (p53, CD8+tumour infiltrating lymphocytes (TILs), mammalian sterile-like 20 kinase 1 (MST1), mucin 2 (MUC2) and urokinase plasminogen activator receptor (uPAR)) and on 208 DLBCL (Bcl2, Bcl6, CD10, FOXP1 and Ki67). The area-under-the-curve (AUC)-over-time plots, cut-off scores for tumour marker positivity and Kaplan–Meier survival curves were analysed.

Results:

With the exception of uPAR, all markers were most accurate within the first 18 months following diagnosis. Expression of p53 (AUC = 0.75), uPAR (AUC = 0.64), Bcl2 (AUC = 0.58) and FOXp1 (AUC = 0.68) was linked to more aggressive tumours, while TILs (AUC = 0.38), MST1 (AUC = 0.39), MUC2 (AUC = 0.38), Bcl6 (AUC = 0.4), CD10 (AUC = 0.49) and Ki67 (AUC = 0.41) were predictive of improved survival. Cut-off scores for markers at their peak accuracies as well as survival time differences were reproducible between colon cancer groups. Only FOXp1 at its optimal cut-off of 60% had significant effects on survival in DLBCL (p = 0.019).

Conclusions:

Time-dependent ROC curve analysis is a novel tool for identifying potential immunohistochemical prognostic markers across varying follow-up times. Use of this tool could facilitate the identification of high-risk patients not only with colon cancer and DLBCL but with a range of other tumour types.
Notes on the St-Narcisse morainic system north of Quebec City
Authors: LaSalle Pierre
Published: October 2008

The Lac St-Charles moraine is interpreted as part of the St-Narcisse moraine system. The stratigraphic position of the morainic sediments suggest that the Lac St-Charles moraine was deposited prior to the highest stand of the Champlain Sea in the Lac St-Charles area.
Geodetic observations for the detection of vertical crustal movement
Authors: Gale L. A.
Published: October 2008

The significant limits within which crustal movement can be determined by geodetic survey methods depend on the quality of the observations, the stability of the markers in the network, and the structure of the network. The paper presents a study of vertical movement in the Lac St. Jean area of Québec. The data used covers the periods 1919–1938 and 1962–1966. With reference to a line of levels from Ste. Anne de la Pérade to La Malbaie along the north shore of the St. Lawrence River, the results show annual uplift as high as 10 mm at Seneterre and subsidence amounting to 4 mm at Stoneham, north of Quebec City. Confidence intervals, at the 95% level, for rates of movement are given as indicators of the quality of the results.
An amendment and extension of tide gauge data analysis for Churchill, Manitoba
Authors: Barnett D. M.
Published: October 2008

An amended rate of land emergence at Churchill, Manitoba is given as 1.3 ft (39 cm) per century with 95% confidence limits of 1.68 ft and 0.85 ft (51.2 and 25.9 cm). It is based on a polynomial regression analysis of tide gauge data for 1940–1968 using the linear function as the most satisfactory.
Some implications of crustal movement in engineering planning
Authors: Clark R. H. , Persoage N. P.
Published: October 2008

Movements of the earth's crust causing progressive changes in the levels of large bodies of water relative to their shorelines may influence the formulation of water resource projects and/or their continuing effectiveness with time. In the Great Lakes basin there is evidence of an uplift of the earth's crust, of about 1 ft per 100 y, in the northeasterly part of the basin relative to that in the southwest. This results in a corresponding lowering of water levels along the northeasterly shorelines and a rise in water levels along the southwest shores. In at least two of the lakes, Lake Huron and Lake Ontario, the average depth of water will change with time. In Lake Huron, it will gradually decrease because the bed underlying the lake is rising with respect to the lake outlet. In Lake Ontario, the depth of water will increase since the lake outlet is rising with respect to the remainder of the lake. This paper reviews some of the engineering implications of the relative rates of crustal movement in the Great Lakes region on long-term management of the water levels of the Great Lakes.
Analysis of pyrrhotite properties accompanying recrystallization of metamorphosed nickel sulfide ore from Kambalda, Western Australia
Authors: Lusk J. , Ostwald J.
Published: October 2008

Serial sectioning was carried out on a single sample of oriented pyrrhotite–pentlandite ore from the Durkin Shoot at Kambalda. This permitted the sequential investigation of a number of pyrrhotite properties including grain morphologies, triple-junction angles, grain lattice orientation, microhardness, peak broadening in X-ray diffractograms, and approximate Fe/S ratios of the monoclinic pyrrhotite by microprobe analysis.The present study confirms the presence of previously identified flattened tabular (F) and equant polyhedral (E) pyrrhotite morphologies, and establishes that the E type has recrystallized from the F variety. The latter is composed of subgrain aggregates with C axes that tend to be oriented perpendicular to the pyrrhotite foliation. Formation of the E type pyrrhotite from the F variety involved subgrain rotations, subgrain coalescence, and grain growth. This was accompanied by definite reductions in σ values for triple-junction angles and in microhardness, as well as apparent reductions in X-ray line broadening and Fe/S ratios for the pyrrhotite. The recrystallization is regarded as a late, low-temperature feature of the regionally metamorphosed nickel sulfide ore.