bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of
Escherichia coli
Strains from Diverse Human and Animal Sources
Authors: Ishii Satoshi , Meyer Katriya P. , Sadowsky Michael J.
Published: July 2007

Escherichia coli
strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with
E. coli
strains present in various animal hosts are not well known. In this study,
E. coli
strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing
E. coli
(STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the
stx
1c
and/or
stx
2d
,
ehxA
, and
saa
genes and belonged to
E. coli
phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked
eae
. In contrast, 26 (1.7%) of the
E. coli
isolates examined were found to be potential enteropathogenic
E. coli
(EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among
E. coli
strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC
ehxA
-positive
E. coli
strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic
E. coli
strains. Our results revealed that potential human health hazards associated with pathogenic
E. coli
strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.
Microbial Isobutyronitrile Utilization under Haloalkaline Conditions
Authors: Sorokin Dimitry Y. , van Pelt Sander , Tourova Tatjana P. , Muyzer Gerard
Published: July 2007

The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus
Marinospirillum
. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two
Bacillus
species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil
Bacillus
grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.
Plutonium(IV) Reduction by the Metal-Reducing Bacteria
Geobacter metallireducens
GS15 and
Shewanella oneidensis
MR1
Authors: Boukhalfa Hakim , Icopini Gary A. , Reilly Sean D. , Neu Mary P.
Published: July 2007

The bacterial reduction of actinides has been suggested as a possible remedial strategy for actinide-contaminated environments, and the bacterial reduction of Pu(VI/V) has the potential to produce highly insoluble Pu(IV) solid phases. However, the behavior of plutonium with regard to bacterial reduction is more complex than for other actinides because it is possible for Pu(IV) to be further reduced to Pu(III), which is relatively more soluble than Pu(IV). This work investigates the ability of the metal-reducing bacteria
Geobacter metallireducens
GS15 and
Shewanella oneidensis
MR1 to enzymatically reduce freshly precipitated amorphous Pu(IV) (OH)
4
[Pu(IV)(OH)
4(am)
] and soluble Pu(IV)(EDTA). In cell suspensions without added complexing ligands, minor Pu(III) production was observed in cultures containing
S. oneidensis
, but little or no Pu(III) production was observed in cultures containing
G. metallireducens
. In the presence of EDTA, most of the Pu(IV)(OH)
4(am)
present was reduced to Pu(III) and remained soluble in cell suspensions of both
S. oneidensis
and
G. metallireducens
. When soluble Pu(IV)(EDTA) was provided as the terminal electron acceptor, cell suspensions of both
S. oneidensis
and
G. metallireducens
rapidly reduced Pu(IV)(EDTA) to Pu(III)(EDTA) with nearly complete reduction within 20 to 40 min, depending on the initial concentration. Neither bacterium was able to use Pu(IV) (in any of the forms used) as a terminal electron acceptor to support growth. These results have significant implications for the potential remediation of plutonium and suggest that strongly reducing environments where complexing ligands are present may produce soluble forms of reduced Pu species.
In Situ Monitoring of the Nascent
Pseudomonas fluorescens
Biofilm Response to Variations in the Dissolved Organic Carbon Level in Low-Nutrient Water by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy
Authors: Delille Anne , Quilès Fabienne , Humbert François
Published: July 2007

Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent
Pseudomonas fluorescens
biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent
P. fluorescens
biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.
Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR
Authors: Vitali Beatrice , Pugliese Ciro , Biagi Elena , Candela Marco , Turroni Silvia , Bellen Gert , Donders Gilbert G. G. , Brigidi Patrizia
Published: July 2007

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and
Lactobacillus
genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by
Lactobacillus
species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of
Lactobacillus
species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.
Genome-Wide Screen for Oxalate-Sensitive Mutants of
Saccharomyces cerevisiae
Authors: Cheng V. , Stotz H. U. , Hippchen K. , Bakalinsky A. T.
Published: July 2007

Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a
Saccharomyces cerevisiae
deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the
rgp1
Δ,
ric1
Δ,
snf7
Δ,
vps16
Δ,
vps20
Δ, and
vps51
Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the
drs2
Δ,
vps16
Δ,
vps51
Δ,
ric1
Δ, and
rib4
Δ mutants, was lethal. With the exception of the
rib4
Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the
rib4
Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.
A Surface-Focused Biotinylation Procedure Identifies the
Yersinia pestis
Catalase KatY as a Membrane-Associated but Non-Surface-Located Protein
Authors: Myers-Morales Tanya , Cowan Clarissa , Gray Michael E. , Wulff Christine R. , Parker Carol E. , Borchers Christoph H. , Straley Susan C.
Published: July 2007

This study identified major surface proteins of the plague bacterium
Yersinia pestis
. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium
Y. pestis
. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by
Y. pestis
cells grown at 37°C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10
9
cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the
Y. pestis
pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.
Serum Lipoproteins Promote Efficient Presentation of the Malaria Virulence Protein PfEMP1 at the Erythrocyte Surface
Authors: Frankland Sarah , Elliott Salenna R. , Yosaatmadja Francisca , Beeson James G. , Rogerson Stephen J. , Adisa Akinola , Tilley Leann
Published: July 2007

The virulence of the malaria parasitePlasmodium falciparumis related to its ability to express a family of adhesive proteins known asP. falciparumerythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.
Energy Metabolism of Bloodstream Form
Trypanosoma theileri
Authors: van Hellemond Jaap J. , Hoek Aad , Schreur Paul Wichgers , Chupin Vladimir , Özdirekcan Suat , Geysen Dirk , van Grinsven Koen W. A. , Koets Ad P. , Van den Bossche Peter , Geerts Stanny , Tielens Aloysius G. M.
Published: July 2007

Bloodstream form
Trypanosoma theileri
degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.
Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase
Authors: Witola William Harold , Ben Mamoun Choukri
Published: July 2007

During its intraerythrocytic life cycle, the malaria parasite
Plasmodium falciparum
undergoes dramatic metabolic and morphological changes and multiplies to produce up to 36 new daughter parasites. This rapid multiplication of the parasite requires an active synthesis of new membranes. The major component of these membranes, phosphatidylcholine, is synthesized via two metabolic routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Here we provide evidence indicating that the activity of the SDPM pathway is regulated by the CDP-choline precursor, choline. We show that the phosphoethanolamine methyltransferase, Pfpmt, a critical enzyme in the SDPM pathway, is down-regulated at the transcriptional level as well as targeted for degradation by the proteasome in the presence of choline. Transcript analysis revealed that Pf
PMT
transcription is repressed by choline in a dose-dependent manner. Immunoblotting, pulse-chase experiments, and immunoprecipitation studies demonstrated that Pfpmt degradation occurs not only in wild-type but also in transgenic parasites constitutively expressing Pfpmt. The proteasome inhibitor bortezomib inhibited choline-mediated Pfpmt degradation. These data provide the first evidence for metabolite-mediated transcriptional and proteasomal regulation in
Plasmodium
and will set the stage for the use of this system for conditional gene and protein expression in this organism.
Evolution of Microcin V and Colicin Ia Plasmids in
Escherichia coli
Authors: Jeziorowski Anne , Gordon David M.
Published: July 2007

Survey results and genotypic characterization of
Escherichia coli
strains demonstrate that the bacteriocins colicin Ia and microcin V coassociate in a strain more often than would be expected by chance. When these two bacteriocins co-occur, they are encoded on the same conjugative plasmid. Plasmids encoding colicin Ia and microcin V are nonrandomly distributed with respect to the genomic background of the host strain. Characterization of microcin V and colicin Ia nucleotide variation, together with the backbone of plasmids encoding these bacteriocins, indicates that the association has evolved on multiple occasions and involves the movement of the microcin V operon, together with the genes
iroNEDCB
and
iss
, onto a nonrandom subset of colicin Ia plasmids. The fitness advantage conferred on cells encoding both colicin Ia and microcin V has yet to be determined.
Insertion Site Occupancy by
stx
2
Bacteriophages Depends on the Locus Availability of the Host Strain Chromosome
Authors: Serra-Moreno Ruth , Jofre Juan , Muniesa Maite
Published: July 2007

Shiga toxin-producing
Escherichia coli
(STEC) is an emergent pathogen characterized by the expression of Shiga toxins, which are encoded in the genomes of lambdoid phages. These phages are infectious for other members of the
Enterobacteriaceae
and establish lysogeny when they integrate into the host chromosome. Five insertion sites, used mainly by these prophages, have been described to date. In the present study, the insertion of
stx
2
prophages in these sites was analyzed in 168 STEC strains isolated from cattle. Additionally, insertion sites were determined for
stx
2
phages which (i) converted diverse laboratory host strains, (ii) coexisted with another
stx
2
prophage, and (iii) infected a recombinant host strain lacking the most commonly used insertion site. Results show that depending on the host strain, phages preferentially use one insertion site. For the most part,
yehV
is occupied in STEC strains while
wrbA
is preferentially selected by the same
stx
phages in
E. coli
laboratory strains. If this primary insertion site is unavailable, then a secondary insertion site is selected. It can be concluded that insertion site occupancy by
stx
phages depends on the host strain and on the availability of the preferred locus in the host strain.
Flagellin Glycans from Two Pathovars of
Pseudomonas syringae
Contain Rhamnose in
d
and
l
Configurations in Different Ratios and Modified 4-Amino-4,6-Dideoxyglucose
Authors: Takeuchi Kasumi , Ono Hiroshi , Yoshida Mitsuru , Ishii Tadashi , Katoh Etsuko , Taguchi Fumiko , Miki Ryuji , Murata Katsuyoshi , Kaku Hanae , Ichinose Yuki
Published: July 2007

Flagellins from
Pseudomonas syringae
pv. glycinea race 4 and
Pseudomonas syringae
pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and
1
H and
13
C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, β-
d
-Qui
p
4N(3-hydroxy-1-oxobutyl)2Me-(1→3)-α-
l
-Rha
p
-(1→2)-α-
l
-Rha
p
. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from
P. syringae
pv. tabaci 6605 consisted solely of
l
-Rha, whereas
P. syringae
pv. glycinea race 4 flagellin contained both
l
-Rha and
d
-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of
P. syringae
.
Structure-Function Analysis of the C-Terminal Domain of LcrV from
Yersinia pestis
Authors: Hamad Mohamad A. , Nilles Matthew L.
Published: July 2007

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in
Yersinia pestis
by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.
The Two-Component Response Regulator RcsB Regulates Type 1 Piliation inEscherichia coli
Authors: Schwan William R. , Shibata Satoshi , Aizawa Shin-Ichi , Wolfe Alan J.
Published: July 2007

The ability ofEscherichia colicells to produce type 1 pili depends upon the orientation of thefimApromoter. The orientation depends upon the ratios of the FimB and FimE recombinases. Here, we report that the two-component response regulator RcsB influences the piliation state by controllingfimBandfimEtranscription.
Cell-to-Cell Heterogeneity in Growth Rate and Gene Expression in
Methylobacterium extorquens
AM1
Authors: Strovas Tim J. , Sauter Linda M. , Guo Xiaofeng , Lidstrom Mary E.
Published: July 2007

Cell-to-cell heterogeneity in gene expression and growth parameters was assessed in the facultative methylotroph
Methylobacterium extorquens
AM1. A transcriptional fusion between a well-characterized methylotrophy promoter (

P
mxaF

) and

gfp
uv

(encoding a variant of green fluorescent protein [GFPuv]) was used to assess single-cell gene expression. Using a flowthrough culture system and laser scanning microscopy, data on fluorescence and cell size were obtained over time through several growth cycles for cells grown on succinate or methanol. Cells were grown continuously with no discernible lag between divisions, and high cell-to-cell variability was observed for cell size at division (2.5-fold range), division time, and growth rate. When individual cells were followed over multiple division cycles, no direct correlation was observed between the growth rate before a division and the subsequent growth rate or between the cell size at division and the subsequent growth rate. The cell-to-cell variability for GFPuv fluorescence from the

P
mxaF

promoter was less, with a range on the order of 1.5-fold. Fluorescence and growth rate were also followed during a carbon shift experiment, in which cells growing on succinate were shifted to methanol. Variability of the response was observed, and the growth rate at the time of the shift from succinate to methanol was a predictor of the response. Higher growth rates at the time of the substrate shift resulted in greater decreases in growth rates immediately after the shift, but full induction of

P
mxaF

-

gfp
uv

was achieved faster. These results demonstrate that in
M. extorquens
, physiological heterogeneity at the single-cell level plays an important role in determining the population response to the metabolic shift examined.
The BclB Glycoprotein of
Bacillus anthracis
Is Involved in Exosporium Integrity
Authors: Thompson Brian M. , Waller Lashanda N. , Fox Karen F. , Fox Alvin , Stewart George C.
Published: July 2007

Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium
Bacillus anthracis
. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of
B. anthracis
are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of
B. anthracis
ΔSterne that carries a deletion of the
bclB
gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A
bclB
mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in
B. anthracis
.
MglA Regulates
Francisella tularensis
subsp.
novicida
(
Francisella novicida
) Response to Starvation and Oxidative Stress
Authors: Guina Tina , Radulovic Dragan , Bahrami Arya J. , Bolton Diana L. , Rohmer Laurence , Jones-Isaac Kendan A. , Chen Jinzy , Gallagher Larry A. , Gallis Byron , Ryu Soyoung , Taylor Greg K. , Brittnacher Mitchell J. , Manoil Colin , Goodlett David R.
Published: July 2007

MglA is a transcriptional regulator of genes that contribute to the virulence of
Francisella tularensis
, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the
F. tularensis
subsp.
novicida
(
F. novicida
) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type
F. novicida
and the
mglA
mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an
F. novicida mglA
mutant were homologs of oxidative and general stress response proteins. The
F. novicida mglA
mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The
F. novicida mglA
mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of
F. tularensis
and is likely essential for bacterial survival in harsh environments.
Global View of the
Clostridium thermocellum
Cellulosome Revealed by Quantitative Proteomic Analysis
Authors: Gold Nicholas D. , Martin Vincent J. J.
Published: July 2007

A metabolic isotope-labeling strategy was used in conjunction with nano-liquid chromatography-electrospray ionization mass spectrometry peptide sequencing to assess quantitative alterations in the expression patterns of subunits within cellulosomes of the cellulolytic bacterium
Clostridium thermocellum
, grown on either cellulose or cellobiose. In total, 41 cellulosomal proteins were detected, including 36 type I dockerin-containing proteins, which count among them all but three of the known docking components and 16 new subunits. All differential expression data were normalized to the scaffoldin CipA such that protein per cellulosome was compared for growth between the two substrates. Proteins that exhibited higher expression in cellulosomes from cellulose-grown cells than in cellobiose-grown cells were the cell surface anchor protein OlpB, exoglucanases CelS and CelK, and the glycoside hydrolase family 9 (GH9) endoglucanase CelJ. Conversely, lower expression in cellulosomes from cells grown on cellulose than on cellobiose was observed for the GH8 endoglucanase CelA; GH5 endoglucanases CelB, CelE, CelG; and hemicellulases XynA, XynC, XynZ, and XghA. GH9 cellulases were the most abundant group of enzymes per CipA when cells were grown on cellulose, while hemicellulases were the most abundant group on cellobiose. The results support the existing theory that expression of scaffoldin-related proteins is coordinately regulated by a catabolite repression type of mechanism, as well as the prior observation that xylanase expression is subject to a growth rate-independent type of regulation. However, concerning transcriptional control of cellulases, which had also been previously shown to be subject to catabolite repression, a novel distinction was observed with respect to endoglucanases.
Uncovering New Metabolic Capabilities of
Bacillus subtilis
Using Phenotype Profiling of Rifampin-Resistant
rpoB
Mutants
Authors: Perkins Amy E. , Nicholson Wayne L.
Published: July 2007

RNA polymerase is a central macromolecular machine controlling the flow of information from genotype to phenotype, and insights into global transcriptional regulation can be gained by studying mutational perturbations in the enzyme. Mutations in the RNA polymerase β subunit gene
rpoB
causing resistance to rifampin (Rif
r
) in
Bacillus subtilis
were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control, such as growth, competence for transformation, sporulation, and germination (H. Maughan, B. Galeano, and W. L. Nicholson, J. Bacteriol. 186:2481-2486, 2004). To better understand the global effects of
rpoB
mutations on metabolism, wild-type and 11 distinct congenic Rif
r
mutant strains of
B. subtilis
were tested for utilization of 95 substrates by use of Biolog GP2 MicroPlates. A number of alterations of substrate utilization patterns were observed in the Rif
r
mutants, including the utilization of novel substrates previously unknown in
B. subtilis
, such as gentiobiose, β-methyl-
d
-glucoside, and
d
-psicose. The results indicate that combining global metabolic profiling with mutations in RNA polymerase provides a system-wide approach for uncovering previously unknown metabolic capabilities and further understanding global transcriptional control circuitry in
B. subtilis
.
The ExpR/Sin Quorum-Sensing System Controls Succinoglycan Production in
Sinorhizobium meliloti
Authors: Glenn Sarah A. , Gurich Nataliya , Feeney Morgan A. , González Juan E.
Published: July 2007

Sinorhizobium meliloti
is a gram-negative soil bacterium capable of forming a symbiotic nitrogen-fixing relationship with its plant host,
Medicago sativa
. Various bacterially produced factors are essential for successful nodulation. For example, at least one of two exopolysaccharides produced by
S. meliloti
(succinoglycan or EPS II) is required for nodule invasion. Both of these polymers are produced in high- and low-molecular-weight (HMW and LMW, respectively) fractions; however, only the LMW forms of either succinoglycan or EPS II are active in nodule invasion. The production of LMW succinoglycan can be generated by direct synthesis or through the depolymerization of HMW products by the action of two specific endoglycanases, ExsH and ExoK. Here, we show that the ExpR/Sin quorum-sensing system in
S. meliloti
is involved in the regulation of genes responsible for succinoglycan biosynthesis as well as in the production of LMW succinoglycan. Therefore, quorum sensing, which has been shown to regulate the production of EPS II, also plays an important role in succinoglycan biosynthesis.
Characterization of Tunisian
Mycobacterium tuberculosis
Rifampin-Resistant Clinical Isolates
Authors: Soudani Alya , Hadjfredj Sondess , Zribi Mériem , Masmoudi Afef , Messaoud Taieb , Tiouri Hanéne , Fendri Chedlia
Published: July 2007

Analysis of the gene encoding the β-subunit of
Mycobacterium tuberculosis
RNA polymerase (
rpoB
) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. In this study, we determined the occurrence of rifampin resistance in 544 Tunisian clinical
M. tuberculosis
strains isolated in a university hospital between 2004 and 2006 by using the standard-proportion agar method, the INNO-LiPA Rif.TB assay, and DNA sequencing.
A FLEXIBLE STRATEGY FOR EMBEDDING AND CONFIGURING RUN-TIME CONTRACT CHECKS IN .NET COMPONENTS
Authors: EDWARDS STEPHEN H. , HAGGARD WESTLEY
Published: July 2007

In component-based systems, there are several obstacles to using Design by Contract (DbC), particularly with respect to third-party components. Contracts are particularly valuable when debugging or testing composite software structures that include third-party components. However, existing approaches have critical weaknesses. First, existing approaches typically require a component's source code to be available if you wish to strip (or re-insert) checks. Second, documentation of the contract is either distributed separately from the component or embedded in the component's source code. Third, enabling and disabling specific kinds of checks on separate components from independent vendors can be a significant challenge. This paper describes an approach to representing contracts for .NET components using attributes. This contract information can be retrieved from the compiled component's metadata and used for many purposes. The paper also describes nContract, a tool that automatically generates run-time checks from embedded contracts. Such run-time checks can be generated and added to a system without requiring source code access or recompilation. Further, when checks for a given component are excluded, they impose no run-time overhead. Finally, a highly expressive, fine-grained mechanism for controlling user preferences about which specific checks are enabled or disabled is presented.
Urokinase-type plasminogen activator and macrophages are required for skeletal muscle hypertrophy in mice
Authors: DiPasquale Dana M. , Cheng Ming , Billich William , Huang Sharon A. , van Rooijen Nico , Hornberger Troy A. , Koh Timothy J.
Published: July 2007

Adult skeletal muscle possesses remarkable potential for growth in response to mechanical loading; however, many of the cellular and molecular mechanisms involved remain undefined. The hypothesis of this study was that the extracellular serine protease, urokinase-type plasminogen activator (uPA), is required for muscle hypertrophy, in part by promoting macrophage accumulation in muscle subjected to increased mechanical loading. Compensatory muscle hypertrophy was induced in mouse plantaris (PLT) muscles by surgical ablation of synergist muscles. Following synergist ablation, PLT muscles in wild-type mice demonstrated edema and infiltration of neutrophils and macrophages but an absence of overt muscle fiber damage. Sham procedures resulted in no edema or accumulation of inflammatory cells. In addition, synergist ablation was associated with a large increase in activity of uPA in the PLT muscle. uPA-null mice demonstrated complete abrogation of compensatory hypertrophy associated with reduced macrophage accumulation, indicating that uPA is required for hypertrophy. Macrophages isolated from wild-type PLT muscle during compensatory hypertrophy expressed uPA and IGF-I, both of which may contribute to hypertrophy. To determine whether macrophages are required for muscle hypertrophy, clodronate liposomes were administered to deplete macrophages in wild-type mice; this resulted in reduced muscle hypertrophy. Decreased macrophage accumulation was associated with reduced cell proliferation but did not alter signaling through the mammalian target of rapamycin pathway. These data indicate that uPA and macrophages are required for muscle hypertrophy following synergist ablation.
Involvement of MMPs in the outward remodeling of collateral mesenteric arteries
Authors: Haas Tara L. , Doyle Jennifer L. , Distasi Matthew R. , Norton Laura E. , Sheridan Kevin M. , Unthank Joseph L.
Published: July 2007

Persistent elevation in shear stress within conduit or resistance arteries causes structural luminal expansion, which serves to normalize shear stress while maintaining increased flow to the downstream vasculature. Although it is known that this adaptation involves cellular proliferation and remodeling of the extracellular matrix, the specific cellular events underlying these responses are poorly understood. Matrix metalloproteinases (MMPs) contribute to extensive remodeling of the extracellular matrix in conduit vessels and vein grafts exposed to high flow. However, involvement of MMPs in remodeling of small muscular collateral arteries, which are exposed to less severe increases in shear stress, has not been tested. We utilized an established model of outward remodeling in mesenteric collateral arteries to determine whether MMPs were upregulated during the remodeling response and to test whether MMP activity was required for luminal expansion. By 4 days, MMP-2 and membrane type 1 MMP (MT1-MMP), but not MMP-9, protein levels were significantly elevated in collateral arteries, as assessed by gelatin zymography and immunostaining. MMP-2 and MT1-MMP proteins, together with their respective transcriptional activators c-Jun and Egr-1 were localized predominantly to the smooth muscle layer of the collateral arteries. The general MMP inhibitor doxycycline prevented luminal expansion of collateral arteries but did not affect the endothelial cell proliferative or medial growth responses. In conclusion, this study provides evidence that MMP-2 and MT1-MMP are upregulated in collateral arteries exposed to elevated shear stress and that MMP activity is essential for the full remodeling response that leads to outward luminal expansion.
Amlodipine inhibits granulation tissue cell apoptosis through reducing calcineurin activity to attenuate postinfarction cardiac remodeling
Authors: Ogino Atsushi , Takemura Genzou , Kanamori Hiromitsu , Okada Hideshi , Maruyama Rumi , Miyata Shusaku , Esaki Masayasu , Nakagawa Munehiro , Aoyama Takuma , Ushikoshi Hiroaki , Kawasaki Masanori , Minatoguchi Shinya , Fujiwara Takako , Fujiwara Hisayoshi
Published: July 2007

Although amlodipine, a long-acting L-type calcium channel blocker, reportedly prevents left ventricular remodeling and dysfunction after myocardial infarction, the mechanism responsible is not yet well understood. Myocardial infarction was induced in mice by ligating the left coronary artery. Treatment of mice with amlodipine (10 mg·kg−1·day−1), beginning on the third day postinfarction, significantly improved survival and attenuated left ventricular dilatation and dysfunction 4 wk postinfarction compared with treatment with saline or hydralazine. Although infarct sizes did not differ among the groups, the infarcted wall thickness was greater and the infarct segment length was smaller in the amlodipine-treated group, and cellular components, including vessels and myofibroblasts, were abundant within the infarcted area. Ten days postinfarction (the subacute stage), the proliferation of granulation tissue cells in the infarcted area was similar among the groups, but the incidence of apoptosis was significantly lower in the amlodipine-treated group, where Bad, a proapoptotic Bcl-2 family protein, was significantly phosphorylated (inactivated). Calcineurin, which dephosphorylates (activates) Bad, was upregulated in infarcted hearts, but its levels were significantly reduced by amlodipine treatment. In vitro, Fas stimulation augmented calcineurin activity and induced apoptosis among infarct tissue-derived myofibroblasts; both of those effects were strongly inhibited by amlodipine, two other calcium channel blockers (verapamil or nifedipine), and two calcineurin inhibitors (cyclosporin A or FK-506). Amlodipine inhibits Fas-mediated granulation tissue cell apoptosis in infarcted hearts, possibly by attenuating the activities of calcineurin and Bad. These findings may provide new insight into the mechanism by which calcium channel blockers attenuate postinfarction cardiac remodeling and dysfunction.
A change in the redox environment and thromboxane A2 production precede endothelial dysfunction in mice
Authors: Gendron Marie-Ève , Thorin Eric
Published: July 2007

We reported that the endothelial dysfunction that develops with age was associated with a proinflammatory phenotype. In this study, we hypothesized that an increased production of proinflammatory cyclooxygenase (COX) products occurs before endothelial dysfunction. Dilations to acetylcholine (ACh) were recorded from pressurized renal arteries isolated from 3- and 6-mo-old C57Bl/6 male mice treated or not with the polyphenol catechin (30 mg·kg−1·day−1) in drinking water for 3 mo. Release of thromboxane (TX) B2, the metabolite of TXA2, was measured by using immunoenzymatic assays, and free radical production was measured by using the fluorescent dye CM-H2DCFDA. Endothelial nitric oxide synthase (eNOS) and COX-1/2 mRNA expression were quantified by quantitative PCR. NG-nitro-l-arginine (l-NNA) reduced ( P < 0.05) ACh-induced dilation in vessels isolated from 3- and 6-mo-old mice. In the presence of l-NNA, indomethacin normalized ( P < 0.05) the dilation in vessels from 6-mo-old mice only. SQ-29548 (PGH2/TXA2 receptor antagonist) and furegrelate (TXA2 synthase inhibitor), in the presence of l-NNA, also improved ( P < 0.05) dilation. l-NNA increased TXA2 release and free radical-associated fluorescence, the latter being prevented by SQ-29548. In vessels from 6-mo-old mice treated with catechin for 3 mo, l-NNA-dependent reduction in ACh-mediated dilation was insensitive to indomethacin, whereas TXA2 release and free radical-associated fluorescence were prevented. eNOS mRNA expression was significantly increased by catechin treatment. Our results suggest that an augmented production of TXA2 and the associated change in redox regulation precede the development of the endothelial dysfunction.
A canine model of septic shock: balancing animal welfare and scientific relevance
Authors: Minneci Peter C. , Deans Katherine J. , Hansen Bernie , Parent Chantal , Romines Chris , Gonzales Denise A. , Ying Sai-Xia , Munson Peter , Suffredini Anthony F. , Feng Jing , Solomon Michael A. , Banks Steven M. , Kern Steven J. , Danner Robert L. , Eichacker Peter Q. , Natanson Charles , Solomon Steven B.
Published: July 2007

A shock canine pneumonia model that permitted relief of discomfort with the use of objective criteria was developed and validated. After intrabronchial Staphylococcus aureus challenge, mechanical ventilation, antibiotics, fluids, vasopressors, sedatives, and analgesics were titrated based on algorithms for 96 h. Increasing S. aureus (1 to 8 × 109colony-forming units/kg) produced decreasing survival rates ( P = 0.04). From 4 to 96 h, changes in arterial-alveolar oxygen gradients, mean pulmonary artery pressure, IL-1, serum sodium levels, mechanical ventilation, and vasopressor support were ordered based on survival time [acute nonsurvivors (≤24 h until death, n = 8) ≥ subacute nonsurvivors (>24 to 96 h until death, n = 8) ≥ survivors (≥96 h until death, n = 22) (all P < 0.05)]. In the first 12 h, increases in lactate and renal abnormalities were greatest in acute nonsurvivors (all P < 0.05). Compared with survivors, subacute nonsurvivors had greater rises in cytokines and liver enzymes and greater falls in platelets, white cell counts, pH, and urine output from 24 to 96 h (all P < 0.05). Importantly, these changes were not attributable to dosages of sedation, which decreased in nonsurvivors [survivors vs. nonsurvivors: 5.0 ± 1.0 vs. 3.8 ± 0.7 ml·h−1·(fentanyl/midazolam/ medetomidine)−1; P = 0.02]. In this model, the pain control regimen did not mask changes in metabolic function and lung injury or the need for more hemodynamic and pulmonary support related to increasing severity of sepsis. The integration into this model of both specific and supportive titrated therapies routinely used in septic patients may provide a more realistic setting to evaluate therapies for sepsis.
Nitric oxide attenuates endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 in vascular smooth muscle cells by a cGMP-dependent pathway
Authors: Bouallegue Ali , Daou Grace Bou , Srivastava Ashok K.
Published: July 2007

Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its antimitogenic and antihypertrophic effect remains unknown. Therefore, the aim of this study was to determine whether NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation, and hypertrophy in A-10 VSMCs. Treatment of A-10 VSMCs with S-nitroso- N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth-promoting and hypertrophic signaling pathways such as ERK1/2, PKB, and Pyk2. On the other hand, inhibition of the endogenous NO generation with NG-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, increased the ET-1-induced phosphorylation of these signaling components. Since NO mediates its effect principally through a cGMP-soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Bromoguanosine 3′,5′-cyclic monophosphate, a nonmetabolizable and cell-permeant analog of cGMP, exhibited a effect similar to that of SNAP and SNP. Furthermore, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.