bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of a Type II Endonuclease (TDE0911) Enables Treponema denticola ATCC 35405 To Accept an Unmethylated Shuttle Vector
Authors: Bian Jiang , Li Chunhao
Published: May 2011

The oral spirochete
Treponema denticola
is associated with human periodontal disease.
T. denticola
ATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared to
T. denticola
ATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence of
T. denticola
ATCC 35405. In this report, we hypothesize that
T. denticola
ATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome of
T. denticola
ATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant of
TDE0911
failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulate
T. denticola
, in particular ATCC 35405, and other strains that may carry similar endonucleases.
Small-Molecule Inhibition of Choline Catabolism in Pseudomonas aeruginosa and Other Aerobic Choline-Catabolizing Bacteria
Authors: Fitzsimmons Liam F. , Flemer Stevenson , Wurthmann A. Sandy , Deker P. Bruce , Sarkar Indra Neil , Wargo Matthew J.
Published: May 2011

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolismin vitroandin situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation inPseudomonas aeruginosa. We used genetic analyses and13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor inP. aeruginosabut did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, includingPseudomonas mendocina,Pseudomonas fluorescens,Pseudomonas putida,Burkholderia cepacia,Burkholderia ambifaria, andSinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolismin situ.
Secretion of Anti-Plasmodium Effector Proteins from a Natural Pantoea agglomerans Isolate by Using PelB and HlyA Secretion Signals
Authors: Bisi Dawn C. , Lampe David J.
Published: May 2011

The insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (
Plasmodium
spp.) in the mosquito host. We hypothesized that
Pantoea agglomerans
, a bacterial symbiont of
Anopheles
mosquitoes, could be engineered to express and secrete anti-
Plasmodium
effector proteins, a strategy termed paratransgenesis. To this end, plasmids that include the
pelB
or
hlyA
secretion signals from the genes of related species (pectate lyase from
Erwinia carotovora
and hemolysin A from
Escherichia coli
, respectively) were created and tested for their efficacy in secreting known anti-
Plasmodium
effector proteins (SM1, anti-Pbs21, and PLA2) in
P. agglomerans
and
E. coli
.
P. agglomerans
successfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-
Plasmodium
activity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.
Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil
Authors: Macdonald Catriona A. , Clark Ian M. , Hirsch Penny R. , Zhao Fang-Jie , McGrath Steve P.
Published: May 2011

Primers were designed to target 16S rRNA and
nodD
genes of
Rhizobium leguminosarum
from DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by both
nodD
and the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional gene
nodD
, providing compelling evidence of a toxic effect of Zn on
R. leguminosarum
populations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH
4
NO
3
-extractable and soil solution Zn.
R. leguminosarum
bv. viciae
nodD
gene copies were generally less sensitive to Zn than
R. leguminosarum
bv. trifolii
nodD.
The latter were generally below detection limits at Zn levels of >250 mg kg
−1
. Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.
Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation
Authors: White Judith , Gilbert Jack , Hill Graham , Hill Edward , Huse Susan M. , Weightman Andrew J. , Mahenthiralingam Eshwar
Published: May 2011

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of
Pseudomonas
(21%),
Burkholderia
(7%), and
Bacillus
(7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed
Proteobacteria
and
Firmicutes
to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by “JW”) was performed,
Betaproteobacteria
(42.8%) and
Gammaproteobacteria
(30.6%) formed the largest proportion of reads; the most abundant genera were
Marinobacter
(15.4%; JW57),
Achromobacter
(41.6%; JW63),
Burkholderia
(80.7%; JW76), and
Halomonas
(66.2%; JW78), all of which were also observed by DGGE. However, the
Clostridia
(38.5%) and
Deltaproteobacteria
(11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a
Pseudomonas
sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a
Mangroveibacter
sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a
Burkholderia
vietnamiensis
strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as
Pseudomonas
.
Patchwork Assembly of
nag
-Like Nitroarene Dioxygenase Genes and the 3-Chlorocatechol Degradation Cluster for Evolution of the 2-Chloronitrobenzene Catabolism Pathway in Pseudomonas stutzeri ZWLR2-1
Authors: Liu Hong , Wang Shu-Jun , Zhang Jun-Jie , Dai Hui , Tang Huiru , Zhou Ning-Yi
Published: May 2011

Pseudomonas stutzeri
ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS
6100
. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from
Burkholderia cepacia
DBO1.
Escherichia coli
cells expressing
cnbAaAbAcAd
converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of
E. coli
/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The
cnbCDEF
gene cluster, homologous to a 3-chlorocatechol degradation cluster in
Sphingomonas
sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the
cnb
cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.
Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification
Authors: Taskin Bilgin , Gozen Ayse Gul , Duran Metin
Published: May 2011

Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with
Escherichia coli
ATCC 25922 as the model organism and the
uidA
gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter
−1
.
Shewanella oneidensis MR-1 Sensory Box Protein Involved in Aerobic and Anoxic Growth
Authors: Sundararajan A. , Kurowski J. , Yan T. , Klingeman D. M. , Joachimiak M. P. , Zhou J. , Naranjo B. , Gralnick J. A. , Fields M. W.
Published: May 2011

Although little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) inShewanella oneidensisMR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of differentc-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, includingc-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia inS. oneidensisMR-1.
Inhibition of the NF-κB Pathway in Human Intestinal Epithelial Cells by Commensal Streptococcus salivarius
Authors: Kaci Ghalia , Lakhdari Omar , Doré Joël , Ehrlich S. Dusko , Renault Pierre , Blottière Hervé M. , Delorme Christine
Published: May 2011

Streptococcus salivarius
exhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8).
Ccr4 Promotes Resolution of the Endoplasmic Reticulum Stress Response during Host Temperature Adaptation in Cryptococcus neoformans
Authors: Havel Virginia E. , Wool Nathan K. , Ayad David , Downey Kurtis M. , Wilson Christabel F. , Larsen Peter , Djordjevic Julianne T. , Panepinto John C.
Published: May 2011

Adaptation to host temperature is a prerequisite for any pathogen capable of causing deep infection in humans. Our previous studies demonstrated that a
Cryptococcus neoformans ccr4
Δ mutant lacking the major deadenylase involved in regulated mRNA decay was defective in host temperature adaptation and therefore virulence. In this study, the
ccr4
Δ mutant was found to exhibit characteristics of chronic unfolded-protein response (UPR) engagement in both the gene expression profile and phenotype. We demonstrate that host temperature adaptation in
C. neoformans
is accompanied by transient induction of the endoplasmic reticulum (ER) stress response and that Ccr4-dependent posttranscriptional gene regulation contributes to resolution of ER stress during host temperature adaptation.
Characterization of the Periplasmic Region of PomB, a Na
+
-Driven Flagellar Stator Protein in Vibrio alginolyticus
Authors: Li Na , Kojima Seiji , Homma Michio
Published: May 2011

The stator proteins PomA and PomB form a complex that couples Na
+
influx to torque generation in the polar flagellar motor of
Vibrio alginolyticus
. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB
C
). To investigate the function of PomB
C
, a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB
C
fragment consisting of residues 135 to 315 (PomB
C5
) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB
ΔL
) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na
+
influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB
ΔL
stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a “plug” segment is important for the control of Na
+
influx through the stator complex as well as for stator assembly.
Modulation of Rho-Dependent Transcription Termination in Escherichia coli by the H-NS Family of Proteins
Authors: Saxena Shivalika , Gowrishankar J.
Published: May 2011

Nascent transcripts in
Escherichia coli
that fail to be simultaneously translated are subject to a factor-dependent mechanism of termination (also termed a polarity) that involves the proteins Rho and NusG. In this study, we found that overexpression of YdgT suppressed the polarity relief phenotypes and restored the efficiency of termination in
rho
or
nusG
mutants. YdgT and Hha belong to the H-NS and StpA family of proteins that repress a large number of genes in Gram-negative bacteria. Variants of H-NS defective in one or the other of its two dimerization domains, but not those defective in DNA binding alone, also conferred a similar suppression phenotype in
rho
and
nusG
mutants. YdgT overexpression was associated with derepression of
proU
, a prototypical H-NS-silenced locus. Polarity relief conferred by
rho
or
nusG
was unaffected in a derivative completely deficient for both H-NS and StpA, although the suppression effects of YdgT or the oligomerization-defective H-NS variants were abolished in this background. Transcription elongation rates
in vivo
were unaffected in any of the suppressor-bearing strains. Finally, the polarity defects of
rho
and
nusG
mutants were exacerbated by Hha and YdgT deficiency. A model is proposed that invokes a novel role for the polymeric architectural scaffold formed on DNA by H-NS and StpA independent of the gene-silencing functions of these nucleoid proteins, in modulating Rho-dependent transcription termination such that interruption of the scaffold (as obtained by expression either of the H-NS oligomerization variants or of YdgT) is associated with improved termination efficiency in the
rho
and
nusG
mutants.
Compromised Factor-Dependent Transcription Termination in a
nusA
Mutant of Escherichia coli: Spectrum of Termination Efficiencies Generated by Perturbations of Rho, NusG, NusA, and H-NS Family Proteins
Authors: Saxena Shivalika , Gowrishankar J.
Published: May 2011

The proteins NusA and NusG, which are essential for the viability of wild-type
Escherichia coli
, participate in various postinitiation steps of transcription including elongation, antitermination, and termination. NusG is required, along with the essential Rho protein, for factor-dependent transcription termination (also referred to as polarity), but the role of NusA is less clear, with conflicting reports that it both promotes and inhibits the process. In this study, we found that a recessive missense
nusA
mutant [
nusA
(
R258C
)] exhibits a transcription termination-defective (that is, polarity-relieved) phenotype, much like missense mutants in
rho
or
nusG
, but is unaffected for either the rate of transcription elongation or antitermination in λ phage. Various combinations of the
rho
,
nusG
, and
nusA
mutations were synthetically lethal, and the lethality was suppressed by expression of the N-terminal half of nucleoid protein H-NS. Our results suggest that NusA function is indeed needed for factor-dependent transcription termination and that an entire spectrum of termination efficiencies can be generated by perturbations of the Rho, NusG, NusA, and H-NS family of proteins, with the corresponding phenotypes extending from polarity through polarity relief to lethality.
Regulon of the
N
-Acetylglucosamine Utilization Regulator NagR in Bacillus subtilis
Authors: Bertram Ralph , Rigali Sébastien , Wood Natalie , Lulko Andrzej T. , Kuipers Oscar P. , Titgemeyer Fritz
Published: May 2011

N
-Acetylglucosamine (GlcNAc) is the most abundant carbon-nitrogen biocompound on earth and has been shown to be an important source of nutrients for both catabolic and anabolic purposes in
Bacillus
species. In this work we show that the GntR family regulator YvoA of
Bacillus subtilis
serves as a negative transcriptional regulator of GlcNAc catabolism gene expression. YvoA represses transcription by binding a 16-bp sequence upstream of
nagP
encoding the GlcNAc-specific EIIBC component of the sugar phosphotransferase system involved in GlcNAc transport and phosphorylation, as well as another very similar 16-bp sequence upstream of the
nagAB-yvoA
locus, wherein
nagA
codes for
N
-acetylglucosamine-6-phosphate deacetylase and
nagB
codes for the glucosamine-6-phosphate (GlcN-6-P) deaminase.
In vitro
experiments demonstrated that GlcN-6-P acts as an inhibitor of YvoA DNA-binding activity, as occurs for its
Streptomyces
ortholog, DasR. Interestingly, we observed that the expression of
nag
genes was still activated upon addition of GlcNAc in a Δ
yvoA
mutant background, suggesting the existence of an auxiliary transcriptional control instance. Initial computational prediction of the YvoA regulon showed a distribution of YvoA binding sites limited to
nag
genes and therefore suggests renaming YvoA to NagR, for
N
-acetylglucosamine utilization regulator. Whole-transcriptome studies showed significant repercussions of
nagR
deletion for several major
B. subtilis
regulators, probably indirectly due to an excess of the crucial molecules acetate, ammonia, and fructose-6-phosphate, resulting from complete hydrolysis of GlcNAc. We discuss a model deduced from NagR-mediated gene expression, which highlights clear connections with pathways for GlcNAc-containing polymer biosynthesis and adaptation to growth under oxygen limitation.
Comparative Genomics of 28 Salmonella enterica Isolates: Evidence for CRISPR-Mediated Adaptive Sublineage Evolution
Authors: Fricke W. Florian , Mammel Mark K. , McDermott Patrick F. , Tartera Carmen , White David G. , LeClerc J. Eugene , Ravel Jacques , Cebula Thomas A.
Published: May 2011

Despite extensive surveillance, food-borne
Salmonella enterica
infections continue to be a significant burden on public health systems worldwide. As the
S. enterica
species comprises sublineages that differ greatly in antigenic representation, virulence, and antimicrobial resistance phenotypes, a better understanding of the species' evolution is critical for the prediction and prevention of future outbreaks. The roles that virulence and resistance phenotype acquisition, exchange, and loss play in the evolution of
S. enterica
sublineages, which to a certain extent are represented by serotypes, remains mostly uncharacterized. Here, we compare 17 newly sequenced and phenotypically characterized nontyphoidal
S. enterica
strains to 11 previously sequenced
S. enterica
genomes to carry out the most comprehensive comparative analysis of this species so far. These phenotypic and genotypic data comparisons in the phylogenetic species context suggest that the evolution of known
S. enterica
sublineages is mediated mostly by two mechanisms, (i) the loss of coding sequences with known metabolic functions, which leads to functional reduction, and (ii) the acquisition of horizontally transferred phage and plasmid DNA, which provides virulence and resistance functions and leads to increasing specialization. Matches between
S. enterica
clustered regularly interspaced short palindromic repeats (CRISPR), part of a defense mechanism against invading plasmid and phage DNA, and plasmid and prophage regions suggest that CRISPR-mediated immunity could control short-term phenotype changes and mediate long-term sublineage evolution. CRISPR analysis could therefore be critical in assessing the evolutionary potential of
S. enterica
sublineages and aid in the prediction and prevention of future
S. enterica
outbreaks.
Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens
Authors: Hibbing Michael E. , Fuqua Clay
Published: May 2011

The plant pathogen
Agrobacterium tumefaciens
encodes predicted iron-responsive regulators, Irr and RirA, that function in several other bacteria to control the response to environmental iron levels. Deletion mutations of
irr
and
rirA
, alone and in combination, were evaluated for their impact on cellular iron response. Growth was severely diminished in the Δ
irr
mutant under iron-limiting conditions, but reversed to wild-type levels in an Δ
irr
Δ
rirA
mutant. The level of uncomplexed iron in the Δ
irr
mutant was decreased, whereas the Δ
rirA
mutant exhibited elevated iron levels. Sensitivity of the Δ
irr
and Δ
rirA
mutants to iron-activated antimicrobial compounds generally reflected their uncomplexed-iron levels. Expression of genes that encode iron uptake systems was decreased in the Δ
irr
mutant, whereas that of iron utilization genes was increased. Irr function required a trihistidine repeat likely to mediate interactions with heme. Iron uptake genes were derepressed in the Δ
rirA
mutant. In the Δ
irr
Δ
rirA
mutant, iron uptake and utilization genes were derepressed, roughly combining the phenotypes of the single mutants. Siderophore production was elevated in the
rirA
mutant, but most strongly regulated by an RirA-controlled sigma factor. Expression of
rirA
itself was regulated by Irr, RirA, and iron availability, in contrast to
irr
expression, which was relatively stable in the different mutants. These studies suggest that in
A. tumefaciens
, the Irr protein is most active under low-iron conditions, inhibiting iron utilization and activating iron acquisition, while the RirA protein is active under high-iron conditions, repressing iron uptake.
Role of Dihydrolipoamide Dehydrogenase in Regulation of Raffinose Transport in Streptococcus pneumoniae
Authors: Tyx Robert E. , Roche-Hakansson Hazeline , Hakansson Anders P.
Published: May 2011

Streptococcus pneumoniae
strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the
raf
operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50- to 80-fold reduction in expression of the
raf
operon genes
aga
(alpha-galactosidase) and
rafEFG
(raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of
raf
operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of
aga
and
rafEFG
, but DLDH did not regulate
rafK
or the putative regulatory genes
rafR
and
rafS
. DLDH also bound directly to RafK both
in vitro
and
in vivo
, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDH-negative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport
in vivo
.
Reliability of Acugraph System for Measuring Skin Conductance at Acupoints
Authors: Mist Scott D , Aickin Mikel , Kalnins Paul , Cleaver Jim , Batchelor Roger , Thorne Tracy , Chamberlin Steve , Tippens Kim , Colbert Agatha P
Published: May 2011

Objective

There are many commercially available instruments for measuring electrical conductance, but there is little information about their reliability. The aim of this study was to quantify measurement variability and assess reliability of the AcuGraph system—a commonly used electrodermal screening device.

Methods

Four experiments were conducted to measure variability in electrical conductance readings obtained by the AcuGraph system. The first involved measuring known resistors. The second measured non-human organic matter. The third was a test–retest assessment of the Yuan-Source and Jing-Well points in 30 healthy volunteers who were measured by a single operator. The fourth was an interoperator reliability evaluation of seven acupuncturists at the Yuan-Source and Jing-Well acupoints on four individuals at two time points.

Results

Against known resistors, the AcuGraph had an average coefficient of variability (CV) of 1.8% between operators and test–retests. On non-human organic material the AcuGraph had an average CV of 0.9% and 2.8%. When a single operator tested 30 participants, the average reliability for the Yuan-Source points was 0.86 and 0.76 for Jing-Well points with a CV of 23.2% and 25.9% respectively. The average CV for the seven acupuncturists was 24.5% on Yuan-Source points and 23.7% on Jing-Well points.

Conclusions

The AcuGraph measures known resistors and organic matter accurately and reliably. Skin conductance at acupoints recorded by one operator was also reliable. There was less consistency in electrodermal recordings obtained by seven different operators. Operator training and technical improvements to the AcuGraph may improve consistency among operators.
Carcinogenic tobacco-specificN-nitrosamines in US cigarettes: three decades of remarkable neglect by the tobacco industry
Authors: Stepanov Irina , Knezevich Aleksandar , Zhang Liqin , Watson Clifford H , Hatsukami Dorothy K , Hecht Stephen S
Published: May 2011

Background

Modification of tobacco curing methods and other changes in cigarette manufacturing techniques could substantially reduce the levels of tobacco-specific nitrosamines (TSNA), a group of potent carcinogens, in cigarette smoke. In 1999, two major US cigarette manufacturers stated their intent to move towards using tobaccos low in TSNA. There is no information available on current TSNA levels in tobacco of various cigarettes available in the US, particularly in the newer varieties introduced over the past decade.

Methods

Seventeen brands of cigarettes were purchased in April of 2010 from retail stores in Minnesota. TSNA levels were measured in the tobacco filler and smoke of these cigarettes.

Results

In all brands, the sum of two potent carcinogenic TSNA - 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone andN'-nitrosonornicotine - in cigarette filler averaged 2.54 (±1.05) μg/g tobacco. This value is virtually identical to the sum of these two carcinogens reported for the tobacco of a US filtered cigarette in 1979. TSNA levels in smoke positively correlated with those in tobacco filler of the same cigarettes.

Conclusion

We found no indication that any meaningful attempt was made to reduce or at least control TSNA levels in the new varieties of the popular brands Marlboro and Camel introduced over the last decade. In light of the recently granted regulatory authority to the FDA over tobacco products, regulation of TSNA levels in cigarette tobacco should be strongly considered to reduce the levels of these potent carcinogens in cigarette smoke.
A GRAPH-BASED APPROACH TO DETECT ABNORMAL SPATIAL POINTS AND REGIONS
Authors: LU CHANG-TIEN , SANTOS RAIMUNDO F. DOS , LIU XUTONG , KOU YUFENG
Published: May 2011

Spatial outliers are the spatial objects whose nonspatial attribute values are quite different from those of their spatial neighbors. Identification of spatial outliers is an important task for data mining researchers and geographers. A number of algorithms have been developed to detect spatial anomalies in meteorological images, transportation systems, and contagious disease data. In this paper, we propose a set of graph-based algorithms to identify spatial outliers. Our method first constructs a graph based on k-nearest neighbor relationship in spatial domain, assigns the differences of nonspatial attribute as edge weights, and continuously cuts high-weight edges to identify isolated points or regions that are much dissimilar to their neighboring objects. The proposed algorithms have three major advantages compared with other existing spatial outlier detection methods: accurate in detecting both point and region outliers, capable of avoiding false outliers, and capable of computing the local outlierness of an object within subgraphs. We present time complexity of the algorithms, and show experiments conducted on US housing and Census data to demonstrate the effectiveness of the proposed approaches.
Evaluation of muscle metaboreflex function through graded reduction in forearm blood flow during rhythmic handgrip exercise in humans
Authors: Ichinose Masashi , Delliaux Stephane , Watanabe Kazuhito , Fujii Naoto , Nishiyasu Takeshi
Published: May 2011

Hypoperfusion of active skeletal muscle elicits a reflex pressor response termed the muscle metaboreflex. Our aim was to determine the muscle metaboreflex threshold and gain in humans by creating an open-loop relationship between active muscle blood flow and hemodynamic responses during a rhythmic handgrip exercise. Eleven healthy subjects performed the exercise at 5 or 15% of maximal voluntary contraction (MVC) in random order. During the exercise, forearm blood flow (FBF), which was continuously measured using Doppler ultrasound, was reduced in five steps by manipulating the inner pressure of an occlusion cuff on the upper arm. The FBF at each level was maintained for 3 min. The initial reductions in FBF elicited no hemodynamic changes, but once FBF fell below a threshold, mean arterial blood pressure (MAP) and heart rate (HR) increased and total vascular conductance (TVC) decreased in a linear manner. The threshold FBF during the 15% MVC trial was significantly higher than during the 5% MVC trial. The gain was then estimated as the slope of the relationship between the hemodynamic responses and FBFs below the threshold. The gains for the MAP and TVC responses did not differ between workloads, but the gain for the HR response was greater in the 15% MVC trial. Our findings thus indicate that increasing the workload shifts the threshold for the muscle metaboreflex to higher blood flows without changing the gain of the reflex for the MAP and TVC responses, whereas it enhances the gain for the HR response.
Angiotensin-converting enzyme inhibition augments the expression of rat elastase-2, an angiotensin II-forming enzyme
Authors: Becari Christiane , Teixeira Felipe R. , Oliveira Eduardo B. , Salgado Maria Cristina O.
Published: May 2011

Mounting evidence suggest that tissue levels of angiotensin (ANG) II are maintained in animals submitted to chronic angiotensin-converting enzyme (ACE) inhibitor treatment. We examined the expression levels of transcripts for elastase-2, a chymostatin-sensitive serine protease identified as the alternative pathway for ANG II generation from ANG I in the rat vascular tissue and the relative role of ACE-dependent and -independent pathways in generating ANG II in the rat isolated carotid artery rings of spontaneously hypertensive rats (SHR) and Wistar normotensive rats (WNR) treated with enalapril for 7 days. Enalapril treatment decreased blood pressure of SHR only and resulted in significantly more elastase-2 mRNA expression in carotid artery of both enalapril-treated WNR and SHR. Captopril induced a comparable rightward shift of concentration-response curves to ANG I in vehicle and enalapril-treated rats, although this effect was of lesser magnitude in SHR group. Chymostatin induced a rightward shift of the dose response to ANG I in vehicle-treated and a decrease in maximal effect of 22% in enalapril-treated WNR group. Maximal response induced by ANG I was remarkably reduced by chymostatin in enalapril-treated SHR carotid artery (by 80%) compared with controls (by 23%). Our data show that chronic ACE inhibition was associated with augmented functional role of non-ACE pathway in generating ANG II and increased elastase-2 gene expression, suggesting that this protease may contribute as an alternative pathway for ANG II generation when ACE is inhibited in the rat vascular tissue.
Epoxyeicosatrienoic acid-dependent cerebral vasodilation evoked by metabotropic glutamate receptor activation in vivo
Authors: Liu Xiaoguang , Li Chunyuan , Gebremedhin Debebe , Hwang Sung Hee , Hammock Bruce D. , Falck John R. , Roman Richard J. , Harder David R. , Koehler Raymond C.
Published: May 2011

Group I metabotropic glutamate receptors (mGluR) on astrocytes have been shown to participate in cerebral vasodilation to neuronal activation in brain slices. Pharmacological stimulation of mGluR in brain slices can produce arteriolar constriction or dilation depending on the initial degree of vascular tone. Here, we examined whether pharmacological stimulation of mGluR in vivo increases cerebral blood flow. A 1-mM solution of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) superfused at 5 μl/min over the cortical surface of anesthetized rats produced a 30 ± 2% (±SE) increase in blood flow measured by laser-Doppler flowmetry after 15–20 min. The response was completely blocked by superfusion of group I mGluR antagonists and attenuated by superfusion of an epoxyeicosatrienoic acid (EET) antagonist (5 ± 4%), an EET synthesis inhibitor (11 ± 3%), and a cyclooxygenase-2 inhibitor (15 ± 3%). The peak blood flow response was not significantly affected by administration of inhibitors of cyclooxygenase-1, neuronal nitric oxide synthase, heme oxygenase, adenosine A2B receptors, or an inhibitor of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE). The blood flow response gradually waned following 30–60 min of DHPG superfusion. This loss of the flow response was attenuated by a 20-HETE synthesis inhibitor and was prevented by superfusion of an inhibitor of epoxide hydrolase, which hydrolyzes EETs. These results indicate that pharmacological stimulation of mGluR in vivo increases cerebral blood flow and that the response depends on the release of EETs and a metabolite of cyclooxygenase-2. Epoxide hydrolase activity and 20-HETE synthesis limit the duration of the response to prolonged mGluR activation.
Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake
Authors: Ginion Audrey , Auquier Julien , Benton Carley R. , Mouton Céline , Vanoverschelde Jean-Louis , Hue Louis , Horman Sandrine , Beauloye Christophe , Bertrand Luc
Published: May 2011

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.
An association between vasomotion and oxygen extraction
Authors: Thorn Clare E. , Kyte Hayley , Slaff Dick W. , Shore Angela C.
Published: May 2011

Vasomotion is defined as a spontaneous local oscillation in vascular tone whose function is unclear but may have a beneficial effect on tissue oxygenation. Optical reflectance spectroscopy and laser Doppler fluximetry provide unique insights into the possible mechanisms of vasomotion in the cutaneous microcirculation through the simultaneous measurement of changes in concentration of oxyhemoglobin ([HbO2]), deoxyhemoglobin ([Hb]), and mean blood saturation (SmbO2) along with blood volume and flux. The effect of vasomotion at frequencies <0.02 Hz attributed to endothelial activity was studied in the dorsal forearm skin of 24 healthy males. Fourier analysis identified periodic fluctuations in SmbO2 in 19 out of 24 subjects, predominantly where skin temperatures were >29.3°C ( X2 = 6.19, P < 0.02). A consistent minimum threshold in SmbO2 (mean: 39.4%, range: 24.0–50.6%) was seen to precede a sudden transient surge in flux, inducing a fast rise in SmbO2. The integral increase in flux correlated with the integral increase in [HbO2] (Pearson's correlation r2 = 0.50, P < 0.001) and with little change in blood volume suggests vasodilation upstream, responding to a low SmbO2 downstream. This transient surge in flux was followed by a sustained period where blood volume and flux remained relatively constant and a steady decrease in [HbO2] and equal and opposite increase in [Hb] was considered to provide a measure of oxygen extraction. A measure of this oxygen extraction has been approximated by the mean half-life of the decay in SmbO2 during this period. A comparison of the mean half-life in the 8 normal subjects [body mass index (BMI) <26.0 kg/m2] of 12.2 s and the 11 obese subjects (BMI >29.5 kg/m2) of 18.8 s was statistically significant (Mann Whitney, P < 0.004). The SmbO2 fluctuated spontaneously in this saw tooth manner by an average of 9.0% (range 4.0–16.2%) from mean SmbO2 values ranging from 30 to 52%. These observations support the hypothesis that red blood cells may act as sensors of local tissue hypoxia, through the oxygenation status of the hemoglobin, and initiate improved local perfusion to the tissue through hypoxic vasodilation.
Synthetic liposomes are protective from bleomycin-induced lung toxicity
Authors: Gwinn William M. , Kapita Mayanga C. , Wang Ping M. , Cesta Mark F. , Martin William J.
Published: May 2011

Idiopathic pulmonary fibrosis is a devastating disease characterized by a progressive, irreversible, and ultimately lethal form of lung fibrosis. Except for lung transplantation, no effective treatment options currently exist. The bleomycin animal model is one of the best studied models of lung injury and fibrosis. A previous study using mouse tumor models observed that liposome-encapsulated bleomycin exhibited reduced lung toxicity. Therefore, we hypothesized that airway delivery of synthetic phosphatidylcholine-containing liposomes alone would protect mice from bleomycin-induced lung toxicity. C57BL/6 mice were administered uncharged multilamellar liposomes (100 μl) or PBS vehicle on day 0 by airway delivery. Bleomycin (3.33 U/kg) or saline vehicle was then given intratracheally on day 1 followed by four additional separate doses of liposomes on days 4, 8, 12, and 16. Fluorescent images of liposomes labeled with 1,1′-dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate confirmed effective and widespread delivery of liposomes to the lower respiratory tract as well as uptake primarily by alveolar macrophages and to a lesser extent by type II alveolar epithelial cells. Results at day 22, 3 wk after bleomycin treatment, showed that airway delivery of liposomes before and after intratracheal administration of bleomycin significantly reduced bleomycin-induced lung toxicity as evidenced by less body weight loss, chronic lung inflammation, and fibrosis as well as improved lung compliance compared with controls. These data indicate that airway-delivered synthetic liposomes represent a novel treatment strategy to reduce the lung toxicity associated with bleomycin in a mouse model.
Tactical Agent Personality
Authors: Tan Chek Tien , Cheng Ho-lun
Published: May 2011

This paper proposes a novel agent personality representation model used to provide interagent adaptation in modern games, coined as the Tactical Agent Personality (TAP). The TAP represents the tactical footprints of a game agent using a weighted network of actions. Directly using the action probabilities to model an agent's personality, removes the time and effort required by experts to craft the model as well as eliminates the performance dependency on expert knowledge. The effectiveness, versatility, generality, scalability, and robustness claims of the TAP architecture and its variations are applied and evaluated across a variety of game scenarios, namely, First-person shooters (FPSs), real-time strategy (RTS) games, and role-playing games (RPG), where they are shown to exhibit plausible adaptive behavior.