bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Characterization of Plasmid-Associated Benzalkonium Chloride Resistance Determinants in a
Listeria monocytogenes
Strain from the 1998-1999 Outbreak
Authors: Elhanafi Driss , Dutta Vikrant , Kathariou Sophia
Published: October 2010

Quaternary ammonium compounds such as benzalkonium chloride (BC) are widely used as disinfectants in both food processing and medical environments. BC-resistant strains of
Listeria monocytogenes
have been implicated in multistate outbreaks of listeriosis and have been frequently isolated from food processing plants. However, the genetic basis for BC resistance in
L. monocytogenes
remains poorly understood. In this study, we have characterized a plasmid (pLM80)-associated BC resistance cassette in
L. monocytogenes
H7550, a strain implicated in the 1998-1999 multistate outbreak involving contaminated hot dogs. The BC resistance cassette (
bcrABC
) restored resistance to BC (MIC, 40 μg/ml) in a plasmid-cured derivative of H7550. All three genes of the cassette were essential for imparting BC resistance. The transcription of H7550 BC resistance genes was increased under sublethal (10 μg/ml) BC exposure and was higher at reduced temperatures (4, 8, or 25°C) than at 37°C. The level of transcription was higher at 10 μg/ml than at 20 or 40 μg/ml.
In silico
analysis suggested that the BC resistance cassette was harbored by an IS
1216
composite transposon along with other genes whose functions are yet to be determined. The findings from this study will further our understanding of the adaptations of this organism to disinfectants such as BC and may contribute to the elucidation of possible BC resistance dissemination in
L. monocytogenes
.
PCR Inhibitor Levels in Concentrates of Biosolid Samples Predicted by a New Method Based on Excitation-Emission Matrix Spectroscopy
Authors: Rock Channah , Alum Absar , Abbaszadegan Morteza
Published: October 2010

Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with glycine-based methods, PCR inhibitors still persist in concentrations that may interfere with nucleic acid amplification. This results in considerable loss of time and resources and increases the probability of false negatives. A method to estimate the degree of inhibition prior to application of molecular methods is desirable. Here we report fluorescence excitation-emission matrix (EEM) profiling as a tool for predicting levels of molecular inhibition in sample concentrates of biosolids.
The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae
Authors: Mallory Michael J. , Law Michael J. , Buckingham Lela E. , Strich Randy
Published: October 2010

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.
Mapping of theNeisseria meningitidisNadA Cell-Binding Site: Relevance of Predicted α-Helices in the NH2-Terminal and Dimeric Coiled-Coil Regions
Authors: Tavano Regina , Capecchi Barbara , Montanari Paolo , Franzoso Susanna , Marin Oriano , Sztukowska Maryta , Cecchini Paola , Segat Daniela , Scarselli Maria , Aricò Beatrice , Papini Emanuele
Published: October 2010

NadA is a trimeric autotransporter protein ofNeisseria meningitidisbelonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup BN. meningitidisstrains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH2-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH2-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH2globular head domain and the NH2dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.
Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in
Mycobacterium smegmatis
Authors: Kurthkoti Krishna , Varshney Umesh
Published: October 2010

Mycobacterium tuberculosis
is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected
M
ycobacterium
smegmatis
, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in
M. smegmatis
by using a hypoxia-responsive promoter of
narK2
from
M. tuberculosis
. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.
Proximity of the Start Codon to a Leaderless mRNA's 5′ Terminus Is a Strong Positive Determinant of Ribosome Binding and Expression in
Escherichia coli
Authors: Krishnan Karthik M. , Van Etten William J. , Janssen Gary R.
Published: October 2010

An AUG start codon is an important determinant of ribosome binding and expression of leaderless mRNAs in
Escherichia coli
. Using reporter constructs encoding mRNAs where the AUG start codon is preceded by untranslated leaders of various length and sequence, we find that close proximity of the start codon to the 5′ terminus and the leader sequence are strong determinants of both ribosome binding and expression.
C-Terminal Domain Residues Important for Secretion and Attachment of RgpB in
Porphyromonas gingivalis
Authors: Slakeski Nada , Seers Christine A. , Ng Kaiting , Moore Caroline , Cleal Steven M. , Veith Paul D. , Lo Alvin W. , Reynolds Eric C.
Published: October 2010

Porphyromonas gingivalis
, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a
P. gingivalis
host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur.
Functional Analysis of Molybdopterin Biosynthesis in Mycobacteria Identifies a Fused Molybdopterin Synthase in
Mycobacterium tuberculosis
Authors: Williams Monique J. , Kana Bavesh D. , Mizrahi Valerie
Published: October 2010

Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the
Mycobacterium tuberculosis
complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of
M. tuberculosis
lacking the
moaA1-moaD1
gene cluster and a derivative in which
moaD2
was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The
M. tuberculosis moaD
and
moaE
homologs were further analyzed by expressing these genes in mutant strains of
M. smegmatis
that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes,
moaD2
and
moaE2
, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of
M. tuberculosis moaD2
in the
M. smegmatis moaD2
mutant and of
M. tuberculosis moaE1
or
moaE2
in the
M. smegmatis moaE2
mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of
M. tuberculosis moaX
also restored MoCo biosynthesis in
M. smegmatis
mutants lacking
moaD2
,
moaE2
, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in
M. tuberculosis
.
Plantazolicin, a Novel Microcin B17/Streptolysin S-Like Natural Product from
Bacillus amyloliquefaciens
FZB42
Authors: Scholz Romy , Molohon Katie J. , Nachtigall Jonny , Vater Joachim , Markley Andrew L. , Süssmuth Roderich D. , Mitchell Douglas A. , Borriss Rainer
Published: October 2010

Here we report on a novel

t

hiazole/

o

xazole-

m

odified

m

icrocin (TOMM) from
Bacillus amyloliquefaciens
FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from
B. amyloliquefaciens
FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ∼10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.
Acis-Encoded Antisense Small RNA Regulated by the HP0165-HP0166 Two-Component System Controls Expression ofureBinHelicobacter pylori
Authors: Wen Yi , Feng Jing , Scott David R. , Marcus Elizabeth A. , Sachs George
Published: October 2010

Expression of urease is essential for gastric colonization byHelicobacter pylori.The increased level of urease in gastric acidity is due, in part, to acid activation of the two-component system (TCS) consisting of the membrane sensor HP0165 and its response regulator, HP0166, which regulates transcription of the seven genes of the urease gene cluster. We now find that there are two majorureABtranscripts: a 2.7-kb full-lengthureABtranscript and a 1.4-kb truncated transcript lacking 3′ureB. Acidic pH (pH 4.5) results in a significant increase in transcription ofureAB, while neutral pH (pH 7.4) increases the truncated 1.4-kb transcript. Northern blot analysis with sense RNA and strand-specific oligonucleotide probes followed by 5′ rapid amplification of cDNA ends detects an antisense small RNA (sRNA) encoded by the 5′ureBnoncoding strand consisting of ∼290 nucleotides (5′ureB-sRNA). Deletion of HP0165 elevates the level of the truncated 1.4-kb transcript along with that of the 5′ureB-sRNA at both pH 7.4 and pH 4.5. Overexpression of 5′ureB-sRNA increases the 1.4-kb transcript, decreases the 2.7-kb transcript, and decreases urease activity. Electrophoretic mobility shift assay shows that unphosphorylated HP0166 binds specifically to the 5′ureB-sRNA promoter. The ability of the HP0165-HP0166 TCS to both increase and decreaseureBexpression at low and high pHs, respectively, facilitates gastric habitation and colonization over the wide range of intragastric pHs experienced by the organism.
The Rcs Signal Transduction Pathway Is Triggered by Enterobacterial Common Antigen Structure Alterations inSerratia marcescens
Authors: Castelli María E. , Véscovi Eleonora García
Published: October 2010

The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to theSerratia marcescensmotile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis ofweccluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of theS. marcescensRcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected inwecmutant strains in other pathogenic bacteria.
The
Burkholderia cenocepacia
LysR-Type Transcriptional Regulator ShvR Influences Expression of Quorum-Sensing, Protease, Type II Secretion, and
afc
Genes
Authors: O'Grady Eoin P. , Nguyen David T. , Weisskopf Laure , Eberl Leo , Sokol Pamela A.
Published: October 2010

Burkholderia cenocepacia
is a significant opportunistic pathogen in individuals with cystic fibrosis. ShvR, a LysR-type transcriptional regulator, has previously been shown to influence colony morphology, biofilm formation, virulence in plant and animal infection models, and some quorum-sensing-dependent phenotypes. In the present study, it was shown that ShvR negatively regulates its own expression, as is typical for LysR-type regulators. The production of quorum-sensing signal molecules was detected earlier in growth in the
shvR
mutant than in the wild type, and ShvR repressed expression of the quorum-sensing regulatory genes
cepIR
and
cciIR
. Microarray analysis and transcriptional fusions revealed that ShvR regulated over 1,000 genes, including the zinc metalloproteases
zmpA
and
zmpB
. The
shvR
mutant displayed increased gene expression of the type II secretion system and significantly increased protease and lipase activities. Both ShvR and CepR influence expression of a 24-kb genomic region adjacent to
shvR
that includes the
afcA
and
afcC
operons, required for the production of an antifungal agent; however, the reduction in expression was substantially greater in the
shvR
mutant than in the
cepR
mutant. Only the
shvR
mutation resulted in reduced antifungal activity against
Rhizoctonia solani.
ShvR, but not CepR, was shown to directly regulate expression of the
afcA
and
afcC
promoters. In summary, ShvR was determined to have a significant influence on the expression of quorum-sensing, protease, lipase, type II secretion, and
afc
genes.
In Vitro
Studies of Peptidoglycan Binding and Hydrolysis by the
Bacillus anthracis
Germination-Specific Lytic Enzyme SleB
Authors: Heffron Jared D. , Sherry Nora , Popham David L.
Published: October 2010

The
Bacillus anthracis
endospore loses resistance properties during germination when its cortex peptidoglycan is degraded by germination-specific lytic enzymes (GSLEs). Although this event normally employs several GSLEs for complete cortex removal, the SleB protein alone can facilitate enough cortex hydrolysis to produce vulnerable spores. As a means to better understand its enzymatic function, SleB was overexpressed, purified, and tested
in vitro
for depolymerization of cortex by measurement of optical density loss and the solubilization of substrate. Its ability to bind peptidoglycan was also investigated. SleB functions independently as a lytic transglycosylase on both intact and fragmented cortex. Most of the muropeptide products that SleB generates are large and are potential substrates for other GSLEs present in the spore. Study of a truncated protein revealed that SleB has two domains. The N-terminal domain is required for stable peptidoglycan binding, while the C-terminal domain is the region of peptidoglycan hydrolytic activity. The C-terminal domain also exhibits dependence on cortex containing muramic-δ-lactam in order to carry out hydrolysis. As the conditions and limitations for SleB activity are further elucidated, they will enable the development of treatments that stimulate premature germination of
B. anthracis
spores, greatly simplifying decontamination measures.
Assembly of the Type II Secretion System: Identification of ExeA Residues Critical for Peptidoglycan Binding and Secretin Multimerization
Authors: Li Gang , Miller Alicia , Bull Harold , Howard S. Peter
Published: October 2010

Aeromonas hydrophilasecretes a number of protein toxins across the outer membrane via the type II secretion system (T2SS). Assembly of the secretion channel ExeD secretin into the outer membrane is dependent on the peptidoglycan binding domain of ExeA. In this study, the peptidoglycan binding domain PF01471 family members were divided into a prokaryotic group and a eukaryotic group. By comparison of their sequence conservation profiles and their representative crystal structures, we found the prokaryotic members to have a highly conserved pocket(s) that is not present in the eukaryotic members. Substitution mutations of nine amino acids of the pocket were constructed in ExeA. Five of the substitution derivatives showed greatly decreased lipase secretion, accompanied by defects in secretin assembly. In addition, usingin vivocross-linking andin vitrocosedimentation assays, we showed that these mutations decreased ExeA-peptidoglycan interactions. These results suggest that the highly conserved pocket in ExeA is the binding site for its peptidoglycan ligand and identify residues critical for this binding.
Insights into the Extracytoplasmic Stress Response ofXanthomonas campestrispv.campestris: Role and Regulation of σE-Dependent Activity
Authors: Bordes Patricia , Lavatine Laure , Phok Kounthéa , Barriot Roland , Boulanger Alice , Castanié-Cornet Marie-Pierre , Déjean Guillaume , Lauber Emmanuelle , Becker Anke , Arlat Matthieu , Gutierrez Claude
Published: October 2010

Xanthomonas campestrispv.campestrisis an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) σ factors. The alternative sigma factor σE, encoded byrpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of σE-dependent activation inX. campestrispv.campestris. We show that therpoEgene is organized as a single transcription unit with the anti-σ generseAand the protease genemucDand thatrpoEtranscription is autoregulated.rseAandmucDtranscription is also controlled by a highly conserved σE-dependent promoter within the σEgene sequence. The σE-mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the σEregulon ofX. campestrispv.campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress σ factor σH. The consensus sequence for the predicted σE-regulated promoter elements is GGAACTN15-17GTCNNA. Determination of therpoHtranscription start site revealed thatrpoHwas directly regulated by σEunder both normal and heat stress conditions. Finally, σEactivity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.
Structural and Functional Analysis of the Type III Secretion System fromPseudomonas fluorescensQ8r1-96
Authors: Mavrodi Dmitri V. , Joe Anna , Mavrodi Olga V. , Hassan Karl A. , Weller David M. , Paulsen Ian T. , Loper Joyce E. , Alfano James R. , Thomashow Linda S.
Published: October 2010

Pseudomonas fluorescensQ8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogenPseudomonas syringae. We also screened a collection of 30 closely relatedP. fluorescensstrains and detected the T3SS genes in all but one of them. The Q8r1-96 genome containedropAAandropMtype III effector genes, which are orthologs of theP. syringaeeffector geneshopAA1-1andhopM1, as well as a novel type III effector gene designatedropB. These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by bothP. syringaeand Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses.
Nucleotide Parasitism by
Simkania negevensis
(
Chlamydiae
)
Authors: Knab Silvia , Mushak Tanja M. , Schmitz-Esser Stephan , Horn Matthias , Haferkamp Ilka
Published: October 2010

Intracellular bacteria live in an environment rich in most essential metabolites but need special mechanisms to access these substrates. Nucleotide transport proteins (NTTs) catalyze the import of ATP and other nucleotides from the eukaryotic host into the bacterial cell and render
de novo
synthesis of these compounds dispensable. The draft genome sequence of
Simkania negevensis
strain Z, a chlamydial organism considered a newly emerging pathogen, revealed four genes encoding putative nucleotide transport proteins (
Sn
NTT1 to
Sn
NTT4), all of which are transcribed during growth of
S. negevensis
in
Acanthamoeba
host cells, as confirmed by reverse transcription-PCR. Using heterologous expression in
Escherichia coli
, we could show that
Sn
NTT1 functions as an ATP/ADP antiporter,
Sn
NTT2 as a guanine nucleotide/ATP/H
+
symporter driven by the membrane potential, and
Sn
NTT3 as a nucleotide triphosphate antiporter. In addition,
Sn
NTT3 is able to transport dCTP, which has not been shown for a prokaryotic transport protein before. No substrate could be identified for
Sn
NTT4. Taking these data together,
S. negevensis
employs a set of nucleotide transport proteins to efficiently tap its host's energy and nucleotide pools. Although similar to other chlamydiae, these transporters show distinct and unique adaptations with respect to substrate specificities and mode of transport.
Mismatch Repair Modulation of MutY Activity Drives
Bacillus subtilis
Stationary-Phase Mutagenesis
Authors: Debora Bernardo N. , Vidales Luz E. , Ramírez Rosario , Ramírez Mariana , Robleto Eduardo A. , Yasbin Ronald E. , Pedraza-Reyes Mario
Published: October 2010

Stress-promoted mutations that occur in nondividing cells (adaptive mutations) have been implicated strongly in causing genetic variability as well as in species survival and evolutionary processes. Oxidative stress-induced DNA damage has been associated with generation of adaptive His
+
and Met
+
but not Leu
+
revertants in strain
Bacillus subtilis
YB955 (
hisC952 metB5 leuC427
). Here we report that an interplay between MutY and MutSL (mismatch repair system [MMR]) plays a pivotal role in the production of adaptive Leu
+
revertants. Essentially, the genetic disruption of MutY dramatically reduced the reversion frequency to the
leu
allele in this model system. Moreover, the increased rate of adaptive Leu
+
revertants produced by a MutSL knockout strain was significantly diminished following
mutY
disruption. Interestingly, although the expression of
mutY
took place during growth and stationary phase and was not under the control of RecA, PerR, or σ
B
, a null mutation in the
mutSL
operon increased the expression of
mutY
several times. Thus, in starved cells, saturation of the MMR system may induce the expression of
mutY
, disturbing the balance between MutY and MMR proteins and aiding in the production of types of mutations detected by reversion to leucine prototrophy. In conclusion, our results support the idea that MMR regulation of the mutagenic/antimutagenic properties of MutY promotes stationary-phase mutagenesis in
B. subtilis
cells.
Mapping Interactions between Germinants and
Clostridium difficile
Spores
Authors: Howerton Amber , Ramirez Norma , Abel-Santos Ernesto
Published: October 2010

Germination of
Clostridium difficile
spores is the first required step in establishing
C. difficile
-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of
C. difficile
spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit
C. difficile
spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The
C. difficile
germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However,
l
-phenylalanine and
l
-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of
C. difficile
spore germination. Similarly,
C. difficile
spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an
m
-aminobenzenesulfonic acid side chain is a strong inhibitor of
C. difficile
spore germination. In conclusion,
C. difficile
spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.
Regulation and Function of
Escherichia coli
Sugar Efflux Transporter A (SetA) during Glucose-Phosphate Stress
Authors: Sun Yan , Vanderpool Carin K.
Published: October 2010

Accumulation of certain nonmetabolizable sugar-phosphates (including α-methyl glucoside-6-phosphate) in
Escherichia coli
is growth inhibitory and elicits the glucose-phosphate stress response. The transcription factor SgrR activates transcription of the small RNA SgrS under stress conditions. SgrS represses translation of mRNAs encoding sugar transporters. The
sgrR
and
sgrS
genes are located directly upstream of
setA
, and this gene organization is conserved in numerous enteric species, prompting the hypothesis that SetA contributes to the glucose-phosphate stress response. SetA is a proton motive force-driven efflux pump capable of transporting various sugars and sugar analogs
in vitro
. This study demonstrates that
setA
expression is induced in response to glucose-phosphate stress, and this requires SgrR. Under stress conditions,
setA
is cotranscribed with
sgrS
from the
sgrS
promoter. A
setA
mutant exhibits a growth defect under stress conditions that can be complemented by
setA
in
trans
, suggesting that SetA contributes to the optimal cellular recovery from stress. Despite previous
in vitro
evidence that SetA can promote efflux of the stress-causing glucose analog α-methyl glucoside,
in vivo
data in this study indicate that SetA is not the major efflux pump responsible for removal of α-methyl glucoside under stress conditions.
The Nodulation of Alfalfa by the Acid-TolerantRhizobiumsp. Strain LPU83 Does Not Require Sulfated Forms of Lipochitooligosaccharide Nodulation Signals
Authors: Torres Tejerizo Gonzalo , Del Papa María Florencia , Soria-Diaz M. Eugenia , Draghi Walter , Lozano Mauricio , Giusti María de los Ángeles , Manyani Hamid , Megías Manuel , Gil Serrano Antonio , Pühler Alfred , Niehaus Karsten , Lagares Antonio , Pistorio Mariano
Published: October 2010

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of thenodgene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-rangeRhizobiumsp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, andLeucena leucocephala. Thenodgene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfa-nodulating rhizobia,Sinorhizobium melilotiandSinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partialnodsequences ofRhizobium mongolenseUSDA 1844 and the corresponding LPU83nodgenes sequences. The phylogenetic analysis of the intergenic regionnodEGpositions strain LPU83 and the type strainR. mongolense1844 in the same branch, which indicates thatRhizobiumsp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from thenodHmutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation.
The Biocide Chlorine Dioxide Stimulates Biofilm Formation inBacillus subtilisby Activation of the Histidine Kinase KinC
Authors: Shemesh Moshe , Kolter Roberto , Losick Richard
Published: October 2010

Bacillus subtilisforms biofilms in response to signals that remain poorly defined. We report that biofilm formation is stimulated by sublethal doses of chlorine dioxide (ClO2), an extremely effective and fast-acting biocide. ClO2accelerated biofilm formation inB. subtilisas well as in other bacteria, suggesting that biofilm formation is a widely conserved response to sublethal doses of the agent. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. We show that the transcription of the major operons responsible for the matrix production inB. subtilis,epsA-epsOandyqxM-sipW-tasA, was enhanced by ClO2, in a manner that depended on the membrane-bound kinase KinC. Activation of KinC appeared to be due to the ability of ClO2to collapse the membrane potential. Importantly, strains unable to make a matrix were hypersensitive to ClO2, indicating that biofilm formation is a defensive response that helps protect cells from the toxic effects of the biocide.
Effects of Deficiency and Overdose of Group 2 Sigma Factors in Triple Inactivation Strains of
Synechocystis
sp. Strain PCC 6803
Authors: Pollari Maija , Rantamäki Susanne , Huokko Tuomas , Kårlund-Marttila Anna , Virjamo Virpi , Tyystjärvi Esa , Tyystjärvi Taina
Published: October 2010

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium
Synechocystis
sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ
70
family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of
Synechocystis
simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the Δ
sigBCD
strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The Δ
sigBCD
, Δ
sigBCE
, and Δ
sigBDE
strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the Δ
sigBCE
strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.
Glucose-Dependent Activation ofBacillus anthracisToxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA
Authors: Chiang Christina , Bongiorni Cristina , Perego Marta
Published: October 2010

Sensing environmental conditions is an essential aspect of bacterial physiology and virulence. InBacillus anthracis, the causative agent of anthrax, transcription of the two major virulence factors, toxin and capsule, is triggered by bicarbonate, a major compound in the mammalian body. Here it is shown that glucose is an additional signaling molecule recognized byB. anthracisfor toxin synthesis. The presence of glucose increased the expression of the protective antigen toxin component-encoding gene (pagA) by stimulating induction of transcription of the AtxA virulence transcription factor. Induction ofatxAtranscription by glucose required the carbon catabolite protein CcpA via an indirect mechanism. CcpA did not bind specifically to any region of the extendedatxApromoter. The virulence of aB. anthracisstrain from which theccpAgene was deleted was significantly attenuated in a mouse model of infection. The data demonstrated that glucose is an important host environment-derived signaling molecule and that CcpA is a molecular link between environmental sensing andB. anthracispathogenesis.
Glypican-3 expression predicts poor clinical outcome of patients with early-stage clear cell carcinoma of the ovary
Authors: Umezu Tomokazu , Shibata Kiyosumi , Kajiyama Hiroaki , Yamamoto Eiko , Nawa Akihiro , Kikkawa Fumitaka
Published: October 2010

Background

Glypican-3 (GPC3), a membrane-bound heparan sulphate proteoglycan, may play a role in promoting cancer cell growth and differentiation. Recent studies reported that GPC3 is overexpressed in clear cell carcinoma (CCC) of the ovary, and not other ovarian histotypes. However, in CCC patients, the relationship between the overexpression of GPC3 and prognosis has not yet been clarified.

Aim

To evaluate GPC3 expression by immunohistochemistry in CCC.

Methods and Results

In 52 CCC patients, GPC3 expression was observed in 40.4%. In cases of CCC, no correlations were identified between GPC3 expression and clinicopathological factors, such as age, FIGO stage, CA125 values, peritoneal cytology, ascitic fluid volume and mortality rate, except for the residual tumour size. GPC3 expression was associated with poor progression-free survival in stage I CCC patients. The numbers of Ki-67-stained cells in GPC3-positive areas were lower than those in GPC3-negative areas. GPC3 expression may be associated with a low proliferation rate in CCC cells. In the early stage of CCC, GPC3-expressing patients tended to be resistant to taxane-based treatment.

Conclusions

Results suggest that the overexpression of GPC3 may be related to the low-level proliferation of tumours; it may be associated with resistance to taxane-based chemotherapy and a poor prognosis in CCC of the ovary.
Management of in situ lobular neoplasia detected on needle core biopsy of breast
Authors: Purdie Colin A , McLean Denis , Stormonth Elizabeth , Macaskill E Jane , McCullough Jean B , Edwards Sharon L , Brown Douglas C , Jordan Lee B
Published: October 2010

Aims

To evaluate the risk of having occult ductal carcinoma in situ or invasive carcinoma in the region of a focus of lobular (in situ) neoplasia (LN) diagnosed on needle core biopsy (NCB) of breast.

Methods

All cases of LN diagnosed on NCB of breast over 10 years (2000–2009 inclusive) were reviewed. The clinical presentation, radiological appearances and final pathological diagnosis on open diagnostic biopsy (ODB) were correlated.

Results

125 cases of LN on NCB were identified from diagnostic codes. Of these, 72 (58%) had a coexistent, higher-grade lesion that mandated surgery. Fifty of the remaining 53 (94%) underwent ODB. The majority of patients were asymptomatic, with 68% presenting through the breast screening programme, and in 89% of patients, the target abnormality was microcalcification. Of the 50 patients, 13 (26%) had a final diagnosis of in situ or invasive carcinoma requiring therapeutic surgery. When the cases of pleomorphic LN were excluded, 21% (10/47) were upgraded. Two of these 10 cases had discordant radiology which could have been diagnosed on repeat NCB leaving an upgrade rate of 18% (8/45). In four of the eight cases of invasive malignancy, the disease was multifocal.

Conclusions

LN is frequently asymptomatic, being identified by mammographic microcalcification alone. In 21% of classical LN cases, it is associated with an undiagnosed, higher-grade lesion requiring oncological management. In our view, patients with LN discovered on NCB should undergo open diagnostic biopsy.
Periprocedural blood pressure management in neurointerventional surgery
Authors: Leslie-Mazwi Thabele M , Sims John R , Hirsch Joshua A , Nogueira Raul G
Published: October 2010

With the increasing range of conditions currently amenable to endovascular therapies, the knowledge of periprocedural blood pressure management is essential for the neurointerventional surgeon. This review discusses the physiology of cerebral blood flow and blood pressure, monitoring options for neurointerventional patients, useful agents for blood pressure elevation and reduction and neuroanesthetic considerations during procedures with an emphasis on practical decision-making. Also included are parameters for conditions typically encountered in the neurointerventional suite based on best available evidence, with reference to blood pressure management before, during and after neurointerventional therapy.
Establishing operational stability—developing human infrastructure
Authors: Gomez Max A , Byers Ernest J , Stingley Preston , Sheridan Robert M , Hirsch Joshua A
Published: October 2010

Over the past year, Toyota has come under harsh scrutiny as a result of several recalls. These well publicized mishaps have not only done damage to Toyota's otherwise sterling reputation for quality but have also called into question the assertions from a phalanx of followers that Toyota's production system (generically referred to as TPS or Lean) is the best method by which to structure one's systems of operation. In this article, we discuss how Toyota, faced with the pressure to grow its business, did not appropriately cadence this growth with the continued development and maintenance of the process capabilities (vis a vis the development of human infrastructure) needed to adequately support that growth. We draw parallels between the pressure Toyota faced to grow its business and the pressure neurointerventional practices face to grow theirs, and offer a methodology to support that growth without sacrificing quality.
A high-fructose diet worsens eccentric left ventricular hypertrophy in experimental volume overload
Authors: Bouchard-Thomassin Andrée-Anne , Lachance Dominic , Drolet Marie-Claude , Couet Jacques , Arsenault Marie
Published: October 2010

The development of left ventricular (LV) hypertrophy (LVH) can be affected by diet manipulation. Concentric LVH resulting from pressure overload can be worsened by feeding rats with a high-fructose diet. Eccentric LVH is a different type of hypertrophy and is associated with volume overload (VO) diseases. The impact of an abnormal diet on the development of eccentric LVH and on ventricular function in chronic VO is unknown. This study therefore examined the effects of a fructose-rich diet on LV eccentric hypertrophy, ventricular function, and myocardial metabolic enzymes in rats with chronic VO caused by severe aortic valve regurgitation (AR). Wistar rats were divided in four groups: sham-operated on control diet (SC; n = 13) or fructose-rich diet (SF; n = 13) and severe aortic regurgitation fed with the same diets [aortic regurgitation on control diet (ARC), n = 16, and aortic regurgitation on fructose-rich diet (ARF), n = 13]. Fructose-rich diet was started 1 wk before surgery, and the animals were euthanized 9 wk later. SF and ARF had high circulating triglycerides. ARC and ARF developed significant LV eccentric hypertrophy after 8 wk as expected. However, ARF developed more LVH than ARC. LV ejection fraction was slightly lower in the ARF compared with ARC. The increased LVH and decreased ejection fraction could not be explained by differences in hemodynamic load. SF, ARC, and ARF had lower phosphorylation levels of the AMP kinase compared with SC. A fructose-rich diet worsened LV eccentric hypertrophy and decreased LV function in a model of chronic VO caused by AR in rats. Normal animals fed the same diet did not develop these abnormalities. Hypertriglyceridemia may play a central role in this phenomenon as well as AMP kinase activity.
Stress-induced opening of the permeability transition pore in the dystrophin-deficient heart is attenuated by acute treatment with sildenafil
Authors: Ascah Alexis , Khairallah Maya , Daussin Frédéric , Bourcier-Lucas Céline , Godin Richard , Allen Bruce G. , Petrof Basil J. , Des Rosiers Christine , Burelle Yan
Published: October 2010

Susceptibility of cardiomyocytes to stress-induced damage has been implicated in the development of cardiomyopathy in Duchenne muscular dystrophy, a disease caused by the lack of the cytoskeletal protein dystrophin in which heart failure is frequent. However, the factors underlying the disease progression are unclear and treatments are limited. Here, we tested the hypothesis of a greater susceptibility to the opening of the mitochondrial permeability transition pore (PTP) in hearts from young dystrophic ( mdx) mice (before the development of overt cardiomyopathy) when subjected to a stress protocol and determined whether the prevention of a PTP opening is involved in the cardioprotective effect of sildenafil, which we have previously reported in mdx mice. Using the 2-deoxy-[3H]glucose method to quantify the PTP opening in ex vivo perfused hearts, we demonstrate that when compared with those of controls, the hearts from young mdx mice subjected to ischemia-reperfusion (I/R) display an excessive PTP opening as well as enhanced activation of cell death signaling, mitochondrial oxidative stress, cardiomyocyte damage, and poorer recovery of contractile function. Functional analyses in permeabilized cardiac fibers from nonischemic hearts revealed that in vitro mitochondria from mdx hearts display normal respiratory function and reactive oxygen species handling, but enhanced Ca2+ uptake velocity and premature opening of the PTP, which may predispose to I/R-induced injury. The administration of a single dose of sildenafil to mdx mice before I/R prevented excessive PTP opening and its downstream consequences and reduced tissue Ca2+ levels. Furthermore, mitochondrial Ca2+ uptake velocity was reduced following sildenafil treatment. In conclusion, beyond our documentation that an increased susceptibility to the opening of the mitochondrial PTP in the mdx heart occurs well before clinical signs of overt cardiomyopathy, our results demonstrate that sildenafil, which is already administered in other pediatric populations and is reported safe and well tolerated, provides efficient protection against this deleterious event, likely by reducing cellular Ca2+ loading and mitochondrial Ca2+ uptake.