bluuebunny/crossref_metadata_2025 huggingface dataset:
The rate of loss of T‐tubules in cultured adult ventricular myocytes is species dependent
Authors: Pavlović Davor , McLatchie Linda M. , Shattock Michael J.
Published: January 2010

In this study, we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72 h culture period. The T‐tubule density was measured in the following two ways: (i) as whole‐cell capacitance in voltage‐clamped myocytes relative to cell area; and (ii) using di‐8‐ANEPPS staining and confocal microscopy. In adult rat ventricular myocytes, whole‐cell capacitance/area was significantly reduced from 47 ± 3 fF μm2 (mean ±s.e.m.; n= 16) in freshly isolated (control) cells to 36 ± 2 fF μm2 (n= 20) after 72 h in culture. The T‐tubular density, as assessed optically using di‐8‐ANEPPS staining, at 48 h was significantly reduced to 70 ± 7% (n= 14) compared with control cells. The T‐tubular density was further reduced after 72 h in culture to 43 ± 7% (n= 10) compared with control cells. In contrast, in mouse myocytes neither whole‐cell capacitance relative to cell area nor optical assessment of T‐tubules showed any significant reduction in capacitance/cell area or T‐tubule density after 72 h of culture. Expression of caveolin‐3 (CAV‐3) (a marker of T‐tubule development) was also measured, and a significant reduction was observed in CAV‐3 expression in rat myocytes at 48 (80 ± 5.5%; n= 6) and 72 h (66 ± 9.5%; n= 6) compared with control cells. The expression of CAV‐3 in mouse myocytes was not significantly reduced even at 72 h. When rat ventricular myocytes were paced in culture for 72 h they exhibited no significant improvement in T‐tubule density or CAV‐3 expression compared with non‐paced cultured cells. In rat myocytes, sarcomere length shortening was significantly reduced in myocytes cultured for 48 (4.96 ± 0.72%; n= 26) and 72 h (4.32 ± 0.80%; n= 26) compared with freshly isolated cells (7.12 ± 0.56%; n= 18). Mouse myocytes, after 24 h in culture, were unable to follow external pacing. These results suggest that detubulation in quiescent culture is slower in the mouse than the rat and that this loss of T‐tubules profoundly affects excitation–contraction coupling in rat myocytes.
On the Kinematics of the Octopus’s Arm
Authors: Levinson Yaron , Segev Reuven
Published: January 2010

The kinematics of the octopus’s arm is studied from the point of view of robotics. A continuum three-dimensional kinematic model of the arm, based on a nonlinear rod theory, is proposed. The model enables the calculation of the strains in various muscle fibers that are required in order to produce a given configuration of the arm—a solution to the inverse kinematics problem. The analysis of the forward kinematics problem shows that the strains in the muscle fibers at two distinct points belonging to a cross section of the arm determine the curvature and the twist of the arm at that cross section. The octopus’s arm lacks a rigid skeleton and the role of material incompressibility in enabling the configuration control is studied.
Shear Induced Removal of Calcium Carbonate Scale From Polypropylene and Copper Tubes
Authors: Royer Matt , Davidson Jane H. , Francis Lorraine F. , Mantell Susan C.
Published: January 2010

This paper presents an analytical model and an experimental study of adhesion and fluid shear removal of calcium carbonate scale on polypropylene and copper tubes in laminar and turbulent water flows, with a view toward understanding how scale can be controlled in solar absorbers and heat exchangers. The tubes are first coated with scale and then inserted in a flow-through apparatus. Removal is measured gravimetrically for Reynolds numbers from 525 to 5550, corresponding to wall shear stresses from 0.16 Pa to 6.0 Pa. The evolutionary structure of the scale is visualized with scanning electron microscopy. Consistent with the predictive model, calcium carbonate is more easily removed from polypropylene than copper. In a laminar flow with a wall shear stress of 0.16 Pa, 65% of the scale is removed from polypropylene while only 10% is removed from copper. Appreciable removal of scale from copper requires higher shear stresses. At Reynolds number of 5500, corresponding to a wall shear stress of 6.0 Pa, 30% of the scale is removed from the copper tubes. The results indicate scale will be more easily removed from polypropylene, and by inference other polymeric materials, than from copper by flushing with water.
640
×
480

pixel
active-matrix Spindt-type field emitter array image sensor with high-gain avalanche rushing amorphous photoconductor target
Authors: Nanba M. , Takiguchi Y. , Honda Y. , Hirano Y. , Watabe T. , Egami N. , Miya K. , Nakamura K. , Taniguchi M. , Itoh S. , Kobayashi A.
Published: January 2010

A 640×480 pixel field emitter array (FEA) image sensor with a high-gain avalanche rushing amorphous photoconductor (HARP) target was fabricated and tested as a step toward the development of ultrahigh-sensitivity compact image sensors for high-definition television cameras. For this sensor, an active-matrix Spindt-type FEA integrated with scanning circuits and a magnetic electron focusing system were designed, and a 15-μm-thick-HARP target was used. The experimental results revealed that the prototype sensor had enough resolution for its pixel number and high sensitivity due to the electrons emitted from the active-matrix FEA being focused onto the HARP target.
Optimization of Pulsed-Field Gel Electrophoresis forLegionella pneumophilaSubtyping
Authors: Zhou Haijian , Ren Hongyu , Zhu Bingqing , Kan Biao , Xu Jianguo , Shao Zhujun
Published: January 2010

A total of 32 strains ofLegionella pneumophilawere used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping ofL. pneumophila. Twenty-six isolates ofL. pneumophilawith various origins and 11 isolates from five different water systems were used as the panels. For optimization of electrophoretic parameters (EPs) of SfiI PFGE, 26 isolates were analyzed with SfiI digestion, using four EPs yielding the sameDvalue. The EP of a switch time of 5 to 50 s for 21 h had the smallest similarity coefficients and was declared the optimal EP for SfiI PFGE ofL. pneumophila. By software analysis and pilot study, AscI was chosen as another PFGE enzyme. AscI PFGE could cluster the isolates from each water system into the same or very similar patterns and had a high degree of typing concordance with other molecular methods. In evaluating the discriminatory power of AscI with the panel of 26 isolates, AscI PFGE gave one single pattern and aDvalue of 100%. AscI PFGE had a high discriminatory power and a high degree of consistency with epidemiological data and other molecular typing methods forL. pneumophilasubtyping, and hence, AscI could be used as a restriction enzyme in PFGE subtyping ofL. pneumophila.
Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese
Authors: Duquenne Manon , Fleurot Isabelle , Aigle Marina , Darrigo Claire , Borezée-Durant Elise , Derzelle Sylviane , Bouix Marielle , Deperrois-Lafarge Véronique , Delacroix-Buchet Agnès
Published: January 2010

Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk.
Staphylococcus aureus
is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during
S. aureus
growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were
ftsZ
,
pta
, and
gyrB
, and these were used as internal controls for RT-qPCR of the genes
sea
and
sed
, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.
Performance Assessment PCR-Based Assays Targeting
Bacteroidales
Genetic Markers of Bovine Fecal Pollution
Authors: Shanks Orin C. , White Karen , Kelty Catherine A. , Hayes Sam , Sivaganesan Mano , Jenkins Michael , Varma Manju , Haugland Richard A.
Published: January 2010

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from
Bacteroidales
. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require
a priori
characterization at each watershed of interest.
Detection of a Reproducible, Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen
Authors: Osborne Catherine A. , Peoples Mark B. , Janssen Peter H.
Published: January 2010

Soil is exposed to hydrogen when symbiotic rhizobia in legume root nodules cannot recycle the hydrogen that is generated during nitrogen fixation. The hydrogen emitted is most likely taken up by free-living soil bacteria that use hydrogen as an energy source, though the bacteria that do this
in situ
remain unclear. In this study, we investigated the effect of hydrogen exposure on the bacteria of two different soils in a microcosm setup designed to simulate hydrogen-emitting root nodules. Although the size and overall composition of the soil bacterial community did not significantly alter after hydrogen exposure, one ribotype increased in relative abundance within each soil. This single-ribotype shift was identified by generating multiple terminal restriction fragment length polymorphism (T-RFLP) profiles of 16S rRNA genes from each soil sample, with gene sequence confirmation to identify terminal restriction fragments. The increased abundance of a single ribotype after hydrogen exposure, within an otherwise similar community, was found in replicate samples taken from each microcosm and was reproducible across replicate experiments. Similarly, only one member of the soil bacterial community increased in abundance in response to hydrogen exposure in soil surrounding the root nodules of field-grown soybean (
Glycine max
). The ribotypes that increased after hydrogen exposure in each soil system tested were all from known hydrogen-oxidizing lineages within the order
Actinomycetales
. We suggest that soil actinomycetes are important utilizers of hydrogen at relevant concentrations in soil and could be key contributors to soil's function as a sink in the global hydrogen cycle.
Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to
Listeria
Phages
Authors: Dupuis Marie-Ève , Moineau Sylvain
Published: January 2010

Virulent phage 1358 is the reference member of a rare group of phages infecting
Lactococcus lactis
. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the
Siphoviridae
family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two
Listeria monocytogenes
phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.
Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination
Authors: Wolf Sandro , Hewitt Joanne , Greening Gail E.
Published: January 2010

Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected humans, pigs, cattle, sheep, deer, and goats, and for the detection of F+ RNA bacteriophage genogroups I to IV, which are associated with human and animal wastes. The sensitivity of this viral toolbox (VTB) was tested against 10-fold dilution series of DNA plasmids that carry the target sequences of the respective viruses and was shown to detect at least 10 plasmid copies for each assay. A panel of human and animal enteric and respiratory viruses showed these assays to be highly sensitive and specific to their respective targets. The VTB was used to detect viruses in fecal and environmental samples, including raw sewage and biosolids from municipal sewage treatment plants, abattoir sewage, and fecally contaminated shellfish and river water, which were likely to contain animal or human viruses.
Growth of
Arthrobacter
sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen
Authors: Husserl Johana , Spain Jim C. , Hughes Joseph B.
Published: January 2010

Arthrobacter
sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.
Transcriptome Analysis of
Escherichia coli
O157:H7 Exposed to Lysates of Lettuce Leaves
Authors: Kyle Jennifer L. , Parker Craig T. , Goudeau Danielle , Brandl Maria T.
Published: January 2010

Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that
Escherichia coli
O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol.
74:
5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157.
Prevalence ofBartonella henselaeandBorrelia burgdorferiSensu Lato DNA inIxodes ricinusTicks in Europe
Authors: Dietrich Florian , Schmidgen Thomas , Maggi Ricardo G. , Richter Dania , Matuschka Franz-Rainer , Vonthein Reinhard , Breitschwerdt Edward B. , Kempf Volkhard A. J.
Published: January 2010

Bartonellaspp. can cause persistent bloodstream infections in humans and animals. To determine whetherBartonella henselaeis present in questingIxodes ricinusticks, we analyzed the prevalence ofB. henselaeDNA among tick stages compared to the prevalence of DNA fromBorrelia burgdorferisensu lato, the pathogen most frequently transmitted by ticks.B. henselaeDNA was present with a prevalence of up to ∼40% in tick populations sampled in four European sites (Eberdingen, Germany; Klasdorf, Germany; Lembach, France; and Madeira, Portugal). The odds of detectingB. henselaeDNA in nymphal ticks was ∼14-fold higher than in adult ticks. No tick was found to be coinfected withB. henselaeandB. burgdorferisensu lato. Taken together, our data indicate that ticks might serve as a vector for the transmission ofB. henselaeto humans.
Galactoxylomannans from Cryptococcus neoformans Varieties
neoformans
and
grubii
Are Structurally and Antigenically Variable
Authors: De Jesus Magdia , Chow Siu-Kei , Cordero Radames J. B. , Frases Susana , Casadevall Arturo
Published: January 2010

Prior studies have established that the
Cryptococcus neoformans
capsular polysaccharide component galactoxylomannan (GalXM) manifests serotype-related structural differences that translate into antigenic differences. We analyzed GalXM from acapsular serotype A and D strains by carbohydrate analysis and static and dynamic light scattering to determine mass, effective diameter, polydispersity, and diffusion coefficients. Multiangle laser light scattering showed that GalXM from
C. neoformans
var.
grubii
strain cap59 (serotype A) had larger molecular mass (4.21 × 10
6
± 0.95 × 10
6
g/mol) and radius of gyration (207 ± 27 nm) than GalXM from
C. neoformans
var.
neoformans
cap67 (serotype D). cap67 GalXM had corresponding values of 0.70 × 10
6
± 0.05 × 10
6
g/mol and 120 ± 22 nm, respectively. The effective diameter for GalXM and polydispersity from the two strains varied depending on temperature and medium growth conditions, indicating that GalXM structure can vary within a strain, depending on its environment. Zeta potential determinations were negative for GalXM from both strains under all conditions, consistent with the recently reported presence of glucuronic acid. These results imply that
C. neoformans
GalXM, like glucuronoxylomannan, can manifest variety- and growth condition-related variations. Analysis of 16
C. neoformans
and 7
Cryptococcus gattii
strains with polyclonal antibody to a GalXM strain revealed antigenic similarities among the
C. neoformans
variety
neoformans
and
grubii
strains and no reactivity with
C. gattii
. As a result of the deleterious effects of GalXM on immune function, structural and antigenic variability between serotypes may translate into differences in immunomodulatory effects.
Global Change of Gene Expression and Cell Physiology in YidC-DepletedEscherichia coli
Authors: Wang Peng , Kuhn Andreas , Dalbey Ross E.
Published: January 2010

YidC depletion affects membrane protein insertion and leads to a defect in the growth of theEscherichia colicell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions using a gene chip method to compare the transcriptomes of JS71 (control) and JS7131 (yidCdepletion strain). Of the more than 4,300 genes identified, 163 were upregulated and 99 were downregulated upon YidC depletion, including genes which are responsible for DNA/RNA repair; energy metabolism; various transporters, proteases and chaperones; stress response; and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found upregulation of the genes encoding the energy transduction proteins F1FoATP synthase and cytochromebo3oxidase due to perturbation in assembly when YidC was depleted. We also determined that the high-level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP), which dissipates the proton motive force. In addition, the gene chip data reveal the Cpx stress pathway is activated upon YidC depletion. The data show the broad physiological contribution of YidC to the bacterial cell and the considerable ramification to the cell when it is depleted.
Characterization of a Thioredoxin-Thioredoxin Reductase System from the Hyperthermophilic BacteriumThermotoga maritima
Authors: Yang Xianqin , Ma Kesen
Published: January 2010

A thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract ofThermotoga maritima. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purifiedT. maritimathioredoxin reductase could not use thioredoxin fromSpirulinaas an electron acceptor, it used thioredoxin that was purified fromT. maritimaby monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparentVmaxvalues of 1,111 ± 35 μmol NADH oxidized min−1mg−1and 115 ± 2.4 μmol NADPH oxidized min−1mg−1, respectively. The apparentKmvalues were determined to be 89 ± 1.1 μM, 73 ± 1.6 μM, and 780 ± 20 μM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95°C, and more than 60% of the activity remained after incubation for 28 h at 80°C. The purifiedT. maritimathioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities.T. maritimathioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5′-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.
The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms inVibrio fischeri
Authors: Bassis Christine M. , Visick Karen L.
Published: January 2010

Bacteria produce different types of biofilms under distinct environmental conditions.Vibrio fischerihas the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the genebinA, located directly downstream ofsyp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squidEuprymna scolopes. Disruption or deletion ofbinAincreased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the ΔbinAmutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion ofbinAincreased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of thesypregulatorsypGinducedbinAexpression. Thus, this work reveals a mechanism by whichV. fischeriinhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.
PepD Participates in the Mycobacterial Stress Response Mediated through MprAB and SigE
Authors: White Mark J. , He Hongjun , Penoske Renee M. , Twining Sally S. , Zahrt Thomas C.
Published: January 2010

Currently, one-third of the world's population is believed to be latently infected with
Mycobacterium tuberculosis
. The mechanisms by which
M. tuberculosis
establishes latent infection remain largely undefined.
mprAB
encodes a two-component signal transduction system required by
M. tuberculosis
for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor
sigE
and the HtrA-like serine protease
pepD
. Recent studies have demonstrated that PepD functions as both a protease and chaperone
in vitro
. In addition, inactivation of
pepD
alters the virulence of
M. tuberculosis
in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of
Mycobacterium
mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317.
pepD
expression initiates from at least three promoters in
M. tuberculosis
, including one that is regulated by SigE and is located upstream of the
mprA
coding sequence. Deletion of
pepD
or
mprAB
in
Mycobacterium smegmatis
and
M. tuberculosis
alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including
sigE
. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.
Modified 3-Oxoadipate Pathway for the Biodegradation of Methylaromatics inPseudomonas reinekeiMT1
Authors: Marín Macarena , Pérez-Pantoja Danilo , Donoso Raul , Wray Victor , González Bernardo , Pieper Dietmar H.
Published: January 2010

Catechols are central intermediates in the metabolism of aromatic compounds. Degradation of 4-methylcatechol via intradiol cleavage usually leads to the formation of 4-methylmuconolactone (4-ML) as a dead-end metabolite. Only a few microorganisms are known to mineralize 4-ML. Themmlgene cluster ofPseudomonasreinekeiMT1, which encodes enzymes involved in the metabolism of 4-ML, is shown here to encode 10 genes found in a 9.4-kb chromosomal region. Reverse transcription assays revealed that these genes form a single operon, where their expression is controlled by two promoters. Promoter fusion assays identified 4-methyl-3-oxoadipate as an inducer. Mineralization of 4-ML is initiated by the 4-methylmuconolactone methylisomerase encoded bymmlI. This reaction produces 3-ML and is followed by a rearrangement of the double bond catalyzed by the methylmuconolactone isomerase encoded bymmlJ. Deletion ofmmlL, encoding a protein of the metallo-β-lactamase superfamily, resulted in a loss of the capability of the strain MT1 to open the lactone ring, suggesting its function as a 4-methyl-3-oxoadipate enol-lactone hydrolase. Further metabolism can be assumed to occur by analogy with reactions known from the 3-oxoadipate pathway.mmlFandmmlGprobably encode a 4-methyl-3-oxoadipyl-coenzyme A (CoA) transferase, and themmlCgene product functions as a thiolase, transforming 4-methyl-3-oxoadipyl-CoA into methylsuccinyl-CoA and acetyl-CoA, as indicated by the accumulation of 4-methyl-3-oxoadipate in the respective deletion mutant. Accumulation of methylsuccinate by anmmlKdeletion mutant indicates that the encoded acetyl-CoA hydrolase/transferase is crucial for channeling methylsuccinate into the central metabolism.
FimR and FimS: Biofilm Formation and Gene Expression inPorphyromonas gingivalis
Authors: Lo Alvin , Seers Christine , Dashper Stuart , Butler Catherine , Walker Glenn , Walsh Katrina , Catmull Deanne , Hoffmann Brigitte , Cleal Steven , Lissel Patricia , Boyce John , Reynolds Eric
Published: January 2010

Porphyromonas gingivalisis a late-colonizing bacterium of the subgingival dental plaque biofilm associated with periodontitis. TwoP. gingivalisgenes,fimRandfimS, are predicted to encode a two-component signal transduction system comprising a response regulator (FimR) and a sensor histidine kinase (FimS). In this study, we show thatfimSandfimR, although contiguous on the genome, are not part of an operon. We inactivatedfimRandfimSin both the afimbriated strain W50 and the fimbriated strain ATCC 33277 and demonstrated that both mutants formed significantly less biofilm than their respective wild-type strains. Quantitative reverse transcription-real-time PCR showed that expression of fimbriation genes was reduced in both thefimSandfimRmutants of strain ATCC 33277. The mutations had no effect, in either strain, on theP. gingivalisgrowth rate or on the response to hydrogen peroxide or growth at pH 9, at 41°C, or at low hemin availability. Transcriptome analysis using DNA microarrays revealed that inactivation offimSresulted in the differential expression of 10% of theP. gingivalisgenome (>1.5-fold;P< 0.05). Notably genes encoding seven different transcriptional regulators, including thefimRgene and three extracytoplasmic sigma factor genes, were differentially expressed in thefimSmutant.
The
Helicobacter pylori
GroES Cochaperonin HspA Functions as a Specialized Nickel Chaperone and Sequestration Protein through Its Unique C-Terminal Extension
Authors: Schauer Kristine , Muller Cécile , Carrière Marie , Labigne Agnès , Cavazza Christine , De Reuse Hilde
Published: January 2010

The transition metal nickel plays a central role in the human gastric pathogen
Helicobacter pylori
because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metal's harmful effects,
H. pylori
is equipped with several specific nickel-binding proteins. Among these,
H. pylori
possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel
in vitro
. To investigate the function of this extension in
H. pylori
, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that
H. pylori
HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.
DsrR, a Novel IscA-Like Protein Lacking Iron- and Fe-S-Binding Functions, Involved in the Regulation of Sulfur Oxidation inAllochromatium vinosum
Authors: Grimm Frauke , Cort John R. , Dahl Christiane
Published: January 2010

In the purple sulfur bacteriumAllochromatium vinosum, the reverse-acting dissimilatory sulfite reductase (DsrAB) is the key enzyme responsible for the oxidation of intracellular sulfur globules. The genesdsrABare the first and the genedsrRis the penultimate of the 15 genes of thedsroperon inA. vinosum. Genes homologous todsrRoccur in a number of other environmentally important sulfur-oxidizing bacteria utilizing Dsr proteins. DsrR exhibits sequence similarities to A-type scaffolds, like IscA, that partake in the maturation of protein-bound iron-sulfur clusters. We used nuclear magnetic resonance (NMR) spectroscopy to solve the solution structure of DsrR and to show that the protein is indeed structurally highly similar to A-type scaffolds. However, DsrR does not retain the Fe-S- or the iron-binding ability of these proteins, which is due to the lack of all three highly conserved cysteine residues of IscA-like scaffolds. Taken together, these findings suggest a common function for DsrR and IscA-like proteins different from direct participation in iron-sulfur cluster maturation. AnA. vinosumΔdsrRdeletion strain showed a significantly reduced sulfur oxidation rate that was fully restored upon complementation withdsrRintrans. Immunoblot analyses revealed a reduced level of DsrE and DsrL in the ΔdsrRstrain. These proteins are absolutely essential for sulfur oxidation. Transcriptional and translational gene fusion experiments suggested the participation of DsrR in the posttranscriptional control of thedsroperon, similar to the alternative function of cyanobacterial IscA as part of the sense and/or response cascade set into action upon iron limitation.
The Three RelE Homologs of
Mycobacterium tuberculosis
Have Individual, Drug-Specific Effects on Bacterial Antibiotic Tolerance
Authors: Singh Ramandeep , Barry Clifton E. , Boshoff Helena I. M.
Published: January 2010

In
Escherichia coli
, expression of the RelE and HipA toxins in the absence of their cognate antitoxins has been associated with generating multidrug-tolerant “persisters.” Here we show that unlike persisters of
E. coli
, persisters of
Mycobacterium tuberculosis
selected with one drug do not acquire cross-resistance to other classes of drugs.
M. tuberculosis
has three homologs of RelE arranged in operons with their apparent antitoxins. Each toxin individually arrests growth of both
M. tuberculosis
and
E. coli
, an effect that is neutralized by coexpression of the cognate antitoxin. Overexpression or deletion of each of the RelE toxins had a toxin- and drug-specific effect on the proportion of bacilli surviving antibiotic killing. All three toxins were upregulated
in vivo
, but none of the deletions affected survival during murine infection. RelE2 overexpression increased bacterial survival rates in the presence of rifampin
in vitro
, while deletion significantly decreased survival rates. Strikingly, deletion of this toxin had no discernible effect on the level of persisters seen in rifampin-treated mice. Our results suggest that,
in vivo
, RelE-generated persisters are unlikely to play a significant role in the generation of bacilli that survive in the face of multidrug therapy or in the generation of multidrug-resistant
M. tuberculosis
.
TnpR Encoded by an IS
Ppu12
Isoform Regulates Transposition of Two Different IS
L3
-Like Insertion Sequences in
Pseudomonas stutzeri
after Conjugative Interaction
Authors: Christie-Oleza J. A. , Nogales B. , Lalucat J. , Bosch R.
Published: January 2010

Pseudomonas stutzeri
AN10 has two IS
L3
-like insertion sequences (ISs). One of them has been recently described as IS
Pst9
. In this study we show that the second IS, situated 4.5 kb upstream of IS
Pst9
, is an isoform of IS
Ppu12
from
Pseudomonas putida
mt-2. Although both IS
L3
-like ISs are flanked by nearly identical (21/24 conserved residues) inverted repeats (IRs) and harbor similar transposases (93% amino acid identity), they differ in their accompanying genes. As described for IS
Pst9
, the isoform of IS
Ppu12
also transposes by a conservative mechanism, forms circular double-stranded DNA (dsDNA) transposition intermediates, and is induced by interaction with the conjugative strain
Escherichia coli
S17-1λ

pir

(conjugative interaction) but not with the nonconjugative
E. coli
DH5α. In fact, we demonstrate that IS
Pst9
transposition after conjugative interaction occurs only when IS
Ppu12
is present, indicating that IS
Ppu12
is upregulating transposition of both ISs under such conditions. We also demonstrate that this conjugative interaction-mediated induction of IS
Ppu12
is not exclusive to the
P. stutzeri
AN10 strain but is a more general phenomenon, at least in
Pseudomonas
. Mutation of TnpR, a MerR-like transcriptional regulator present in IS
Ppu12
but not in IS
Pst9
, reduced the transcription of
tnpA
(IS
Ppu12
transposase-encoding gene) and decreased formation of circular dsDNA transposition intermediates after conjugative interaction. Complementation of the TnpR mutant restored the phenotype. In addition, the presence of TnpR in an IS
Ppu12
-free genetic background did not induce IS
Pst9
after conjugative interaction. Thus, our results suggest that TnpR, after conjugative interaction, activates transcription of
tnpA
of IS
Ppu12
. Then, TnpA of IS
Ppu12
would bind to IRs of both ISs, IS
Ppu12
and IS
Pst9
, causing their transposition.
In
Helicobacter pylori
, LuxS Is a Key Enzyme in Cysteine Provision through a Reverse Transsulfuration Pathway
Authors: Doherty Neil C. , Shen Feifei , Halliday Nigel M. , Barrett David A. , Hardie Kim R. , Winzer Klaus , Atherton John C.
Published: January 2010

In many bacteria, LuxS functions as a quorum-sensing molecule synthase. However, it also has a second, more central metabolic function in the activated methyl cycle (AMC), which generates the
S
-adenosylmethionine required by methyltransferases and recycles the product via methionine.
Helicobacter pylori
lacks an enzyme catalyzing homocysteine-to-methionine conversion, rendering the AMC incomplete and thus making any metabolic role of
H. pylori
LuxS (LuxS
Hp
) unclear. Interestingly,
luxS
Hp
is located next to genes annotated as
cysK
Hp
and
metB
Hp
, involved in other bacteria in cysteine and methionine metabolism. We showed that isogenic strains carrying mutations in
luxS
Hp
,
cysK
Hp
, and
metB
Hp
could not grow without added cysteine (whereas the wild type could), suggesting roles in cysteine synthesis. Growth of the Δ
luxS
Hp
mutant was restored by homocysteine or cystathionine and growth of the Δ
cysK
Hp
mutant by cystathionine only. The Δ
metB
Hp
mutant had an absolute requirement for cysteine. Metabolite analyses showed that
S
-ribosylhomocysteine accumulated in the Δ
luxS
Hp
mutant, homocysteine in the Δ
cysK
Hp
mutant, and cystathionine in the Δ
metB
Hp
mutant. This suggests that
S
-ribosylhomocysteine is converted by LuxS
Hp
to homocysteine (as in the classic AMC) and thence by CysK
Hp
to cystathionine and by MetB
Hp
to cysteine.
In silico
analysis suggested that
cysK-metB-luxS
were acquired by
H. pylori
from a Gram-positive source. We conclude that
cysK-metB-luxS
encode the capacity to generate cysteine from products of the incomplete AMC of
H. pylori
in a process of reverse transsulfuration. We recommend that the misnamed genes
cysK
Hp
and
metB
Hp
be renamed
mccA
(methionine-to-cysteine-conversion gene A) and
mccB
, respectively.
Tricarboxylic Acid Cycle-Dependent Synthesis of
Staphylococcus aureus
Type 5 and 8 Capsular Polysaccharides
Authors: Sadykov Marat R. , Mattes Theodoric A. , Luong Thanh T. , Zhu Yefei , Day Shandra R. , Sifri Costi D. , Lee Chia Y. , Somerville Greg A.
Published: January 2010

Staphylococcus aureus
capsule synthesis requires the precursor
N
-acetyl-glucosamine; however, capsule is synthesized during post-exponential growth when the availability of
N
-acetyl-glucosamine is limited. Capsule biosynthesis also requires aerobic respiration, leading us to hypothesize that capsule synthesis requires tricarboxylic acid cycle intermediates. Consistent with this hypothesis,
S. aureus
tricarboxylic acid cycle mutants fail to make capsule.
The Chemoreceptor Dimer Is the Unit of Conformational Coupling and Transmembrane Signaling
Authors: Amin Divya N. , Hazelbauer Gerald L.
Published: January 2010

Transmembrane chemoreceptors are central components in bacterial chemotaxis. Receptors couple ligand binding and adaptational modification to receptor conformation in processes that create transmembrane signaling. Homodimers, the fundamental receptor structural units, associate in trimers and localize in patches of thousands. To what degree do conformational coupling and transmembrane signaling require higher-order interactions among dimers? To what degree are they altered by such interactions? To what degree are they inherent features of homodimers? We addressed these questions using nanodiscs to create membrane environments in which receptor dimers had few or no potential interaction partners. Receptors with many, few, or no interaction partners were tested for conformational changes and transmembrane signaling in response to ligand occupancy and adaptational modification. Conformation was assayed by measuring initial rates of receptor methylation, a parameter independent of receptor-receptor interactions. Coupling of ligand occupancy and adaptational modification to receptor conformation and thus to transmembrane signaling occurred with essentially the same sensitivity and magnitude in isolated dimers as for dimers with many neighbors. Thus, we conclude that the chemoreceptor dimer is the fundamental unit of conformational coupling and transmembrane signaling. This implies that in signaling complexes, coupling and transmembrane signaling occur through individual dimers and that changes between dimers in a receptor trimer or among trimer-based signaling complexes are subsequent steps in signaling.
Complete Genome Sequence ofListeria seeligeri, a Nonpathogenic Member of the GenusListeria
Authors: Steinweg Christiane , Kuenne Carsten T. , Billion André , Mraheil Mobarak A. , Domann Eugen , Ghai Rohit , Barbuddhe Sukhadeo B. , Kärst Uwe , Goesmann Alexander , Pühler Alfred , Weisshaar Bernd , Wehland Jürgen , Lampidis Robert , Kreft Jürgen , Goebel Werner , Chakraborty Trinad , Hain Torsten
Published: January 2010

We report the complete and annotated genome sequence of the nonpathogenicListeria seeligeriSLCC3954 serovar 1/2b type strain harboring the smallest completely sequenced genome of the genusListeria.
Positions of Trp Codons in the Leader Peptide-Coding Region of the
at
Operon Influence Anti-Trap Synthesis and
trp
Operon Expression in
Bacillus licheniformis
Authors: Levitin Anastasia , Yanofsky Charles
Published: January 2010

Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In
Bacillus subtilis
and some other Gram-positive bacteria,
trp
operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNA
Trp
. Tryptophan-activated TRAP binds to
trp
operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In
B. subtilis
, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNA
Trp
. In this paper, we focus on explaining the differences in organization and regulatory functions of the
at
operon's leader peptide-coding region,
rtpLP
, of
B. subtilis
and
Bacillus licheniformis
. Our objective was to correlate the greater growth sensitivity of
B. licheniformis
to tryptophan starvation with the spacing of the three Trp codons in its
at
operon leader peptide-coding region. Our findings suggest that the Trp codon location in
rtpLP
of
B. licheniformis
is designed to allow a mild charged-tRNA
Trp
deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis.
Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate inPetrotoga mobilis
Authors: Fernandes Chantal , Mendes Vitor , Costa Joana , Empadinhas Nuno , Jorge Carla , Lamosa Pedro , Santos Helena , da Costa Milton S.
Published: January 2010

The compatible solute mannosylglucosylglycerate (MGG), recently identified inPetrotoga miotherma, also accumulates inPetrotoga mobilisin response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome ofPtg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome ofPtg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose andd-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme fromPtg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene fromThermotoga maritimawas successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG inPtg. mobilis.