bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Protection against Paraquat‐induced Oxidative Damage by Abscisic Acid in Maize Leaves is Mediated through Mitogen‐activated Protein Kinase
Authors: Ding Hai‐Dong , Zhang Xiao‐Hua , Xu Shu‐Cheng , Sun Li‐Li , Jiang Ming‐Yi , Zhang A‐Ying , Jin Yin‐Gen
Published: September 2009

Mitogen‐activated protein kinase (MAPK) cascade has been shown to be important components in stress signal transduction pathway. In the present study, protection of maize seedlings (Zea mays L.) against paraquat‐generated oxidative toxicity by abscisic acid (ABA), its association with MAPK and ZmMPK5, a candidate for MAPK were investigated. Treatment of maize leaves with exogenous ABA led to significant decreases in the content of malondialdehyde, the percentage of ion leakage and the level of protein oxidation (in terms of carbonyl groups) under paraquat (PQ) stress. However, such decreases were blocked by the pretreatment with two MAPK kinase inhibitors PD98059 and U0126. The damage caused by PQ was further aggravated by inhibitors. Two inhibitors also suppressed the total activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). Besides, treatment with PQ stimulated the activation of a 46 kDa MAPK, which was identified as ZmMPK5 by in‐gel kinase assay with immunoprecipitation. These results reveal that ABA‐induced protection against PQ‐generated oxidative damage is mediated through MAPK cascade in maize leaves, in which ZmMPK5, a candidate for MAPK, is demonstrated to be involved.
Differential Neonatal and Postneonatal Infant Mortality Rates Across US Counties: The Role of Socioeconomic Conditions and Rurality
Authors: Sparks P. Johnelle , McLaughlin Diane K. , Stokes C. Shannon
Published: September 2009

ABSTRACT: 
Purpose:

To examine differences in correlates of neonatal and postneonatal infant mortality rates, across counties, by degree of rurality. Methods: Neonatal and postneonatal mortality rates were calculated from the 1998 to 2002 Compressed Mortality Files from the National Center for Health Statistics. Bivariate analyses assessed the relationship between neonatal and postneonatal mortality by Urban Influence (UI) codes. Multivariable, weighted least‐squares regression models included measures of county socioeconomic conditions, health services and environmental risks. Findings: The bivariate analysis indicated neonatal and postneonatal mortality was significantly higher in the most nonmetropolitan counties compared to the most metropolitan counties. However the relationship was not linear across the Urban Influence codes. In the multivariable models, a nonmetropolitan advantage was observed for counties not adjacent to metropolitan areas for neonatal mortality. However, postneonatal mortality rates were higher in the most rural nonmetropolitan counties. Conclusions: Certain characteristics of nonmetropolitan counties not adjacent to metropolitan counties and with an urban area of 2,500 population or more are protective against neonatal mortality (UI = 7, UI = 8). This may indicate that just having access to health services is more important to creating a protective effect for these nonmetropolitan counties than having a high concentration of medical facilities. The nonmetropolitan, not adjacent (UI = 9) disadvantage observed for postneonatal mortality supports the idea that the isolation of these areas combined with the combination of risk factors across the most nonmetropolitan counties leads to poorer postneonatal health outcomes in these areas.
Non‐quantal release of acetylcholine from parasympathetic nerve terminals in the right atrium of rats
Authors: Abramochkin Denis V. , Nurullin Leniz F. , Borodinova Anastasia A. , Tarasova Nadezhda V. , Sukhova Galina S. , Nikolsky Eugen E. , Rosenshtraukh Leonid V.
Published: September 2009

Acetylcholinesterase (AChE) inhibitors provoke typical cholinergic effects in the isolated right atrium of the rat due to the accumulation of acetylcholine (ACh). Our study was designed to show that in the absence of vagal impulse activity, ACh is released from the parasympathetic nerve fibres by means of non‐quantal secretion. The conventional microelectrode technique was used to study changes in action potential (AP) configuration in the right atrium preparation of rats during application of AChE inhibitors. Staining with the lipophilic fluorescent dye FM1‐43 was used to demonstrate the presence of endocytosis in cholinergic endings. The AChE inhibitors armin (10−7–10−5 m) and neostigmine (10−7to 5 × 10−6 m) caused a reduction of AP duration and prolonged the cycle length. These effects were abolished by atropine and were therefore mediated by ACh accumulated in the myocardium during AChE inhibition. Putative block of impulse activity of the postganglionic neurons by tetrodotoxin (5 × 10−7 m) and blockade of ganglionic transmission by hexomethonium (2 × 10−4 m), as well as blockade of all forms of quantal release withClostridium botulinumtype A toxin (50 U ml−1), did not alter the effects of armin. Experiments with FM1‐43 dye confirmed the effective block of exocytosis by botulinum toxin. Selective inhibition of the choline uptake system using hemicholinium III (10−5 m), which blocks non‐quantal release at the neuromuscular junction, suppressed the effects of AChE inhibitors. Thus, accumulation of ACh is likely to be caused by non‐quantal release from cholinergic terminals. We propose that non‐quantal release of ACh, shown previously at the neuromuscular junction, is present in cholinergic postganglionic fibres of the rat heart in addition to quantal release.
Quantification of the Relative Effects of Temperature, pH, and Water Activity on Inactivation of
Escherichia coli
in Fermented Meat by Meta-Analysis
Authors: McQuestin Olivia J. , Shadbolt Craig T. , Ross Tom
Published: September 2009

Outbreaks of
Escherichia coli
infections linked to fermented meats have prompted much research into the kinetics of
E. coli
inactivation during fermented meat manufacture. A meta-analysis of data from 44 independent studies was undertaken that allowed the relative influences of pH, water activity (a
w
), and temperature on
E. coli
survival during fermented meat processing to be investigated. Data were reevaluated to determine rates of inactivation, providing 484 rate data points with various pH (2.8 to 6.14), a
w
(0.75 to 0.986), and temperature (−20 to 66°C) values, product formulations, and
E. coli
strains and serotypes. When the data were presented as an Arrhenius model, temperature (0 to 47°C) accounted for 61% of the variance in the ln(inactivation rate) data. In contrast, the pH or a
w
measured accounted for less than 8% of variability in the data, and the effects of other pH- and a
w
-based variables (i.e., total decrease and rates of reduction of those factors) were largely dependent on the temperature of the process. These findings indicate that although temperatures typically used in fermented meat manufacture are not lethal to
E. coli
per se, when other factors prevent
E. coli
growth (e.g., low pH and a
w
), the rate of inactivation of
E. coli
is dominated by temperature. In contrast, inactivation rates at temperatures above ∼50°C were characterized by smaller
z
values than those at 0 to 47°C, suggesting that the mechanisms of inactivation are different in these temperature ranges. The Arrhenius model developed can be used to improve product safety by quantifying the effects of changes in temperature and/or time on
E. coli
inactivation during fermented meat manufacture.
Quadruplex Real-Time PCR Assay for Detection and Identification of
Vibrio cholerae
O1 and O139 Strains and Determination of Their Toxigenic Potential
Authors: Huang Jianwei , Zhu Yumei , Wen Huixin , Zhang Jiafeng , Huang Shijie , Niu Jianjun , Li Qingge
Published: September 2009

Vibrio cholerae
is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (
ctxA
), the hemolysin gene (
hlyA
), O1-specific
rfb
, and O139-specific
rfb
, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of
V. cholerae
and 31 strains of non-
V. cholerae
organisms. The analytical sensitivity for detection of toxigenic
V. cholerae
O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify
V. cholerae
and reliably predict the toxigenic potential of strains detected.
Complex Phenotypic and Genotypic Responses of
Listeria monocytogenes
Strains Exposed to the Class IIa Bacteriocin Sakacin P
Authors: Tessema Girum Tadesse , Møretrø Trond , Kohler Achim , Axelsson Lars , Naterstad Kristine
Published: September 2009

Sakacin P is a class IIa bacteriocin that is active against the food-borne pathogen
Listeria monocytogenes
, and use of this compound as a biopreservative in foods has been suggested. In the present study, we characterized 30 spontaneous sakacin P-resistant mutants of
L. monocytogenes
obtained after single exposure to sakacin P. The frequency of development of sakacin P resistance for all strains was in the range from 10
−8
to 10
−9
. Using the 50% inhibitory concentration (IC
50
) of sakacin P, the strains could be grouped into strains with high levels of resistance (IC
50
, ≥10
4
ng ml
−1
) and strains with low levels of resistance (IC
50
, <10
4
ng ml
−1
). Resistant strains belonging to the same IC
50
group also had similar physiological and genetic characteristics. Generally, the resistant strains showed substantial variations in many parameters, such as differences in the stability of the acquired resistance to sakacin P, growth fitness, food-related stress tolerance, and biofilm-forming ability. Fourier transform infrared spectroscopy revealed differences between wild-type and resistant strains in polysaccharide, fatty acid, and, protein regions. A mannose-specific phosphotransferase (PTS) operon has been described for class IIa bacteriocin resistance, and the sakacin P-resistant strains displayed both up- and downregulation of the expression of the
mptA
gene encoding the PTS system. This is the first comprehensive study of the diversity of a large number of spontaneous resistant mutants obtained after one exposure to a class IIa bacteriocin, particularly to sakacin P. The great diversity among the resistant strains exposed to the same stress conditions suggests that there are different resistance mechanisms.
Identification of
Bacillus anthracis
by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks
Authors: Lasch Peter , Beyer Wolfgang , Nattermann Herbert , Stämmler Maren , Siegbrecht Enrico , Grunow Roland , Naumann Dieter
Published: September 2009

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax,
Bacillus anthracis. Bacillus
cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from
Bacillus
and related genera, among them 102 strains of
B. anthracis
and 121 strains of
B. cereus
. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate
t
tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent
t
tests suggested
B. anthracis
- and
B. cereus
group-specific signals. For example, mass spectra of
B. anthracis
exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus
Bacillus
. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of
B. anthracis
, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.
Comparison of Point-of-Use Technologies for Emergency Disinfection of Sewage-Contaminated Drinking Water
Authors: McLennan S. Devin , Peterson Lauren A. , Rose Joan B.
Published: September 2009

Four point-of-use disinfection technologies for treating sewage-contaminated well water were compared. Three systems, based on flocculant-disinfectant packets and
N
-halamine chlorine and bromine contact disinfectants, provided a range of 4.0 to >6.6 log
10
reductions (LR) of naturally occurring fecal indicator and heterotrophic bacteria and a range of 0.9 to >1.9 LR of coliphage.
Development of a Log-Quadratic Model To Describe Microbial Inactivation, Illustrated by Thermal Inactivation of
Clostridium botulinum
Authors: Stone G. , Chapman B. , Lovell D.
Published: September 2009

In the commercial food industry, demonstration of microbiological safety and thermal process equivalence often involves a mathematical framework that assumes log-linear inactivation kinetics and invokes concepts of decimal reduction time (

D
T

),
z
values, and accumulated lethality. However, many microbes, particularly spores, exhibit inactivation kinetics that are not log linear. This has led to alternative modeling approaches, such as the biphasic and Weibull models, that relax strong log-linear assumptions. Using a statistical framework, we developed a novel log-quadratic model, which approximates the biphasic and Weibull models and provides additional physiological interpretability. As a statistical linear model, the log-quadratic model is relatively simple to fit and straightforwardly provides confidence intervals for its fitted values. It allows a

D
T

-like value to be derived, even from data that exhibit obvious “tailing.” We also showed how existing models of non-log-linear microbial inactivation, such as the Weibull model, can fit into a statistical linear model framework that dramatically simplifies their solution. We applied the log-quadratic model to thermal inactivation data for the spore-forming bacterium
Clostridium botulinum
and evaluated its merits compared with those of popular previously described approaches. The log-quadratic model was used as the basis of a secondary model that can capture the dependence of microbial inactivation kinetics on temperature. This model, in turn, was linked to models of spore inactivation of Sapru et al. and Rodriguez et al. that posit different physiological states for spores within a population. We believe that the log-quadratic model provides a useful framework in which to test vitalistic and mechanistic hypotheses of inactivation by thermal and other processes.
Identification and Classification ofbclGenes and Proteins ofBacillus cereusGroup Organisms and Their Application inBacillus anthracisDetection and Fingerprinting
Authors: Leski Tomasz A. , Caswell Clayton C. , Pawlowski Marcin , Klinke David J. , Bujnicki Janusz M. , Hart Sean J. , Lukomski Slawomir
Published: September 2009

TheBacillus cereusgroup includes three closely related species,B. anthracis,B. cereus, andB. thuringiensis, which form a highly homogeneous subdivision of the genusBacillus. One of these species,B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of theBacilluscollagen-like proteinbclgenes as a basis forB. anthracisdetection and fingerprinting. Five genes, designatedbclAtobclE, are present inB. anthracisstrains. Examination ofbclABCDEsequences identified polymorphisms inbclBalleles of theB. cereusgroup organisms. These sequence polymorphisms allowed specific detection ofB. anthracisstrains by PCR using both genomic DNA and purifiedBacillusspores in reactions. By exploiting the length variation of thebclalleles it was demonstrated that the combinedbclABCDEPCR products generate markedly different fingerprints for theB. anthracisAmes and Sterne strains. Moreover, we predict thatbclABCDElength polymorphism creates unique signatures forB. anthracisstrains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept forB. anthracisdetection and fingerprinting, which can be used alone or in combination with previously established typing platforms.
Novel GH10 Xylanase, with a Fibronectin Type 3 Domain, from
Cellulosimicrobium
sp. Strain HY-13, a Bacterium in the Gut of
Eisenia fetida
Authors: Kim Do Young , Han Mi Kyoung , Park Doo-Sang , Lee Jong Suk , Oh Hyun-Woo , Shin Dong-Ha , Jeong Tae-Sook , Kim Sung Uk , Bae Kyung Sook , Son Kwang-Hee , Park Ho-Yong
Published: September 2009

The gene encoding a novel modular xylanase from
Cellulosimicrobium
sp. strain HY-13 was identified and expressed in
Escherichia coli
, and its truncated gene product was characterized. The enzyme consisted of three distinct functional domains, an N-terminal catalytic GH10 domain, a fibronectin type 3 domain, and C-terminal carbohydrate-binding module 2.
Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus
Authors: Kilaru Sreedhar , Collins Catherine M. , Hartley Amanda J. , Bailey Andy M. , Foster Gary D.
Published: September 2009

We describe efficient polyethylene glycol (PEG)-mediated andAgrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus,Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation ofC. passeckerianus. The PEG-mediated transformation ofC. passeckerianusprotoplasts was successful and generated hygromycin-resistant transformants more efficiently than either phleomycin or carboxin resistance.Agrobacterium-mediated transformation with plasmid pBGgHg containinghphgene under the control of theAgaricus bisporus gpdIIpromoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for theAgrobacterium-mediated system within the genome.C. passeckerianusactin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression inC. passeckerianus, but only when constructs contained a 5′ intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated usinggfpas a target gene easily scored inC. passeckerianus. Upon transformation ofgfpantisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression inC. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.
Bacteria
,
Archaea
, and
Crenarchaeota
in the Epilimnion and Hypolimnion of a Deep Holo-Oligomictic Lake
Authors: Callieri Cristiana , Corno Gianluca , Caravati Emanuele , Rasconi Serena , Contesini Mario , Bertoni Roberto
Published: September 2009

In a deep, subalpine holo-oligomictic lake, the relative abundance of
Archaea
and
Crenarchaeota
, but not that of
Bacteria
, increases significantly with depth and varies seasonally. Cell-specific prokaryotic productivity is homogeneous along the water column. The concept of active
Archaea
observed in the deep ocean can therefore be extended to a deep oxic lake.
Molecular Identification and Hidden Diversity of Novel
Daphnia
Parasites from European Lakes
Authors: Wolinska Justyna , Giessler Sabine , Koerner Henrike
Published: September 2009

Parasites play important roles in local population dynamics and genetic structure. However, due to insufficient diagnostic tools, detailed host-parasite interactions may remain concealed by hidden parasite diversity in natural systems. Microscopic examination of 19 European lake
Daphnia
populations revealed the presence of three groups of parasites: fungi, microsporidia, and oomycetes. For most of these parasites no genetic markers have been described so far. Based on sequence similarities of the nuclear small-subunit and internal transcribed spacer (ITS) rRNA gene regions, one fungus, four microsporidian, and nine oomycete taxa were discovered in 147 infected
Daphnia
(and/or three other zooplankton crustaceans). Additionally, cloning of rRNA gene regions revealed parasite sequence variation within host individuals. This was most pronounced in the ITS region of one microsporidian taxon, where the within-host sequence variation ranged from 1.7% to 5.3% polymorphic sites for parasite isolates from 14 different geographical locations. Interestingly, the parasite isolates from close locations grouped together based on sequence similarities, suggesting that there was parasite dispersal. Taken together, the data obtained in this study revealed hidden diversity of parasite communities in
Daphnia
lake populations. Moreover, a higher level of resolution for identifying parasite strains makes it possible to test new hypotheses with respect to parasite dispersal, transmission routes, and coinfection.
Listeria monocytogenes
Attachment to and Detachment from Stainless Steel Surfaces in a Simulated Dairy Processing Environment
Authors: Poimenidou Sofia , Belessi Charalambia A. , Giaouris Efstathios D. , Gounadaki Antonia S. , Nychas George-John E. , Skandamis Panagiotis N.
Published: September 2009

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of
Listeria monocytogenes
on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (10
7
CFU/ml or CFU/g) and incubated at two temperatures (5 and 20°C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5°C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5°C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20°C). The opposite was observed when custard incubated at 20°C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while
L. monocytogenes
was not detectable on SS coupons after 7 days of incubation (at 5°C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5°C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached
L. monocytogenes
cells.
Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from
Streptomyces peucetius
ATCC 27952 in
Escherichia coli
Authors: Ghimire Gopal Prasad , Lee Hei Chan , Sohng Jae Kyung
Published: September 2009

Putative hopanoid genes from
Streptomyces peucetius
were introduced into
Escherichia coli
to improve the production of squalene, an industrially important compound. High expression of
hopA
and
hopB
(encoding squalene/phytoene synthases) together with
hopD
(encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of
dxs
and
idi
,
E. coli
genes encoding 1-deoxy-
d
-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.
Microbial Populations Responsive to Denitrification-Inducing Conditions in Rice Paddy Soil, as Revealed by Comparative 16S rRNA Gene Analysis
Authors: Ishii Satoshi , Yamamoto Michihiro , Kikuchi Mami , Oshima Kenshiro , Hattori Masahira , Otsuka Shigeto , Senoo Keishi
Published: September 2009

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum
Firmicutes
, including a unique
Symbiobacterium
clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders
Burkholderiales
and
Rhodocyclales
, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus
Herbaspirillum
and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to
Herbaspirillum
are potential key players in nitrate respiration and denitrification under the conditions used.
ATP-Dependent but Proton Gradient-Independent Polyphosphate-Synthesizing Activity in Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus
Authors: Tani Chiharu , Ohtomo Ryo , Osaki Mitsuru , Kuga Yukari , Ezawa Tatsuhiro
Published: September 2009

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas.
Glomus
sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A
1
or a protonophore, suggesting that ATP may not energize the reaction through H
+
-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.
Thermostable Cyanuric Acid Hydrolase from
Moorella thermoacetica
ATCC 39073
Authors: Li Qingyan , Seffernick Jennifer L. , Sadowsky Michael J. , Wackett Lawrence P.
Published: September 2009

Cyanuric acid, a metabolic intermediate in the degradation of many
s
-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile
Moorella thermoacetica
ATCC 39073 was cloned, expressed in
Escherichia coli
, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and
N
-methylisocyanuric acid. The
M. thermoacetica
enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from
M. thermoacetica
is the most promising enzyme to use for cyanuric acid remediation applications.
Immortalized Human Brain Endothelial Cell Line HCMEC/D3 as a Model of the Blood-Brain Barrier Facilitates In Vitro Studies of Central Nervous System Infection by
Cryptococcus neoformans
Authors: Vu Kiem , Weksler Babette , Romero Ignacio , Couraud Pierre-Olivier , Gelli Angie
Published: September 2009

Cryptococcus neoformans
cells must cross the blood-brain barrier prior to invading the central nervous system. Here we demonstrate that the immortalized human brain endothelial cell line HCMEC/D3 is a useful alternative to primary brain endothelial cells as a model of the blood-brain barrier for studies of central nervous system infection.
Zinc-Independent Folate Biosynthesis: Genetic, Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB
Authors: Sankaran Banumathi , Bonnett Shilah A. , Shah Kinjal , Gabriel Scott , Reddy Robert , Schimmel Paul , Rodionov Dmitry A. , de Crécy-Lagard Valérie , Helmann John D. , Iwata-Reuyl Dirk , Swairjo Manal A.
Published: September 2009

GTP cyclohydrolase I (GCYH-I) is an essential Zn
2+
-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in
Bacteria
and
Archaea
, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g.,
Bacillus subtilis
, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn
2+
-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn
2+
starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn
2+
-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.
Thioredoxin 1 Participates in the Activity of theSalmonella entericaSerovar Typhimurium Pathogenicity Island 2 Type III Secretion System
Authors: Negrea Aurel , Bjur Eva , Puiac Speranta , Ygberg Sofia Eriksson , Åslund Fredrik , Rhen Mikael
Published: September 2009

The facultative intracellular pathogenSalmonella entericaserovar Typhimurium relies on itsSalmonellapathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in atrxAmutant. Complementation analyses using wild-typetrxAor a genetically engineeredtrxAcoding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.
A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC
Authors: Pilonieta M. Carolina , Erickson Kimberly D. , Ernst Robert K. , Detweiler Corrella S.
Published: September 2009

Antimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs.Salmonella entericadeploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. TheydeIgene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here thatydeIis additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS).ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates thatompDcontributes to polymyxin B resistance, and bothydeIandompDare important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs.
A 5′ Leader Sequence Regulates Expression of Methanosarcinal CO Dehydrogenase/Acetyl Coenzyme A Synthase
Authors: Anderson Kimberly L. , Apolinario Ethel E. , MacAuley Sheridan R. , Sowers Kevin R.
Published: September 2009

In vivo expression of CO dehydrogenase/acetyl coenzyme A synthase in
Methanosarcina
spp. is coordinately regulated in response to substrate by at least two mechanisms: differential transcription initiation and early elongation termination near the 3′ end of a 371-bp leader sequence. This is the first report of regulation of transcription elongation in the
Archaea
.
Functional Analysis of theHelicobacter pyloriFlagellar Switch Proteins
Authors: Lowenthal Andrew C. , Hill Marla , Sycuro Laura K. , Mehmood Khalid , Salama Nina R. , Ottemann Karen M.
Published: September 2009

Helicobacter pyloriuses flagellum-mediated chemotaxis to promote infection. Bacterial flagella change rotational direction by changing the state of the flagellar motor via a subcomplex referred to as the switch. Intriguingly, theH. pylorigenome encodes four switch complex proteins, FliM, FliN, FliY, and FliG, instead of the more typical three ofEscherichia coliorBacillus subtilis. Our goal was to examine whether and how all four switch proteins participate in flagellation. Previous work determined that FliG was required for flagellation, and we extend those findings to show that all four switch proteins are necessary for normal numbers of flagellated cells. Furthermore, whilefliYandfliNare partially redundant with each other, both are needed for wild-type levels of flagellation. We also report the isolation of anH. pyloristrain containing an R54C substitution infliM, resulting in bacteria that swim constantly and do not change direction. Along with data demonstrating that CheY-phosphate interacts with FliM, these findings suggest that FliM functions inH. pylorimuch as it does in other organisms.
Structural and Genetic Basis for the Serological Differentiation ofPasteurella multocidaHeddleston Serotypes 2 and 5
Authors: St. Michael Frank , Harper Marina , Parnas Henrietta , John Marietta , Stupak Jacek , Vinogradov Evgeny , Adler Ben , Boyce John D. , Cox Andrew D.
Published: September 2009

Pasteurella multocidais classified into 16 serotypes according to the Heddleston typing scheme. As part of a comprehensive study to define the structural and genetic basis of this scheme, we have determined the structure of the lipopolysaccharide (LPS) produced byP. multocidastrains M1404 (B:2) and P1702 (E:5), the type strains for serotypes 2 and 5, respectively. The only difference between the LPS structures made by these two strains was the absence of a phosphoethanolamine (PEtn) moiety at the 3 position of the second heptose (Hep II) in M1404. Analysis of thelpt-3gene, required for the addition of this PEtn residue, revealed that the gene was intact in P1702 but contained a nonsense mutation in M1404. Expression of an intact copy oflpt-3in M1404 resulted in the attachment of a PEtn residue to the 3 position of the Hep II residue, generating an LPS structure identical to that produced by P1702. We identified and characterized each of the glycosyltransferase genes required for assembly of the serotype 2 and 5 LPS outer core. Monoclonal antibodies raised against serotype 2 LPS recognized the serotype 2/5-specific outer core LPS structure, but recognition of this structure was inhibited by the PEtn residue on Hep II. These data indicate that the serological classification of strains into Heddleston serotypes 2 and 5 is dependent on the presence or absence of PEtn on Hep II.
Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by
Salmonella
Pathogenicity Island 2
Authors: Miki Tsuyoshi , Shibagaki Yoshio , Danbara Hirofumi , Okada Nobuhiko
Published: September 2009

The type III secretion system (T3SS) encoded by
Salmonella
pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of
Salmonella enterica
serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An
S. enterica
serovar Typhimurium
ssaE
mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an
sseA
deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast,
ssaE
mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an
ssaE
(
I55G
) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for
Salmonella
virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.
ThedltOperon ofBacillus cereusIs Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects
Authors: Abi Khattar Z. , Rejasse A. , Destoumieux-Garzón D. , Escoubas J. M. , Sanchis V. , Lereclus D. , Givaudan A. , Kallassy M. , Nielsen-Leroux C. , Gaudriault S.
Published: September 2009

Thedltoperon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of thedltoperon in insect virulence by inactivating this operon inBacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The ΔdltBcmutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B fromSpodoptera frugiperda. ΔdltBcwas as pathogenic as the wild-type strain in oral infections ofGalleria mellonellabut much less virulent when injected into the hemocoels ofG. mellonellaandSpodoptera littoralis. We detected thedltoperon in three gram-negative genera:Erwinia(Erwinia carotovora),Bordetella(Bordetella pertussis,Bordetella parapertussis, andBordetella bronchiseptica), andPhotorhabdus(the entomopathogenic bacteriumPhotorhabdus luminescensTT01, thedltoperon of which did not restore cationic AMP resistance in ΔdltBc). We suggest that thedltoperon protectsB. cereusagainst insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.
Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface
Authors: Mihajlovic Sanja , Lang Silvia , Sut Marta V. , Strohmaier Heimo , Gruber Christian J. , Koraimann Günther , Cabezón Elena , Moncalián Gabriel , de la Cruz Fernando , Zechner Ellen L.
Published: September 2009

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of
oriT
plasmid DNA in vivo (Δ

L
k

= −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at
nic
but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.
Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1
Authors: Sut Marta V. , Mihajlovic Sanja , Lang Silvia , Gruber Christian J. , Zechner Ellen L.
Published: September 2009

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at
nic
. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.