bluuebunny/crossref_metadata_2025 huggingface dataset:
Asymptotic Analytical Solutions of First-Passage Rate to Quasi-Nonintegrable Hamiltonian Systems
Authors: Lin Deng Mao , Fu Yue , Long Huang Zhi
Published: May 2014

The first-passage problem of quasi-nonintegrable Hamiltonian systems subject to light linear/nonlinear dampings and weak external/parametric random excitations is investigated here. The motivation is to acquire asymptotic analytical solution of the first-passage rate or the mean first-passage time based on the averaged Itô stochastic differential equation for quasi-nonintegrable Hamiltonian systems. By using the probability current equation and the Laplace integral method, a new method is proposed to obtain the asymptotic analytical expressions for the first-passage rate in the case of high passage threshold. The associated functions such as the reliability function and the probability density function of first-passage time can then be obtained from the first-passage rate. High passage threshold is the crucial condition for the validity of the proposed method. The random bistable oscillator is studied as an illustrative example using the method. The analytical result obtained from the asymptotic analysis shows its consistency with the Kramers formula. A coupled two-degree-of-freedom (2DOF) nonlinear oscillator subjected to stochastic excitations is studied to illustrate the procedure of acquiring the asymptotic analytical solution. The results obtained from the analytical solution agree well with those from numerical simulation, which verifies the accuracy of the proposed method.
Uptake Coefficients of Some Volatile Organic Compounds by Soot and Their Application in Understanding Particulate Matter Evolution in Aircraft Engine Exhaust Plumes
Authors: Yu Zhenhong , Liscinsky David S. , True Bruce , Peck Jay , Jennings Archer C. , Wong Hsi-Wu , Jun Mina , Franklin Jonathan , Herndon Scott C. , Waitz Ian A. , Miake-Lye Richard C.
Published: May 2014

To assist microphysical modeling on particulate matter (PM) evolution emitted from aircraft engines, uptake coefficients of some volatile organic compounds on soot were experimentally determined in this study. The determined values vary from (1.0 ± 0.1) × 10−6 for water-miscible propylene glycol to (2.5 ± 0.1) × 10−5 for 2,6-dimethylnaphthalene, a polycyclic aromatic hydrocarbon. An inverse power-law correlation between uptake coefficient on soot and solubility in water was observed. Using the correlation, microphysical simulations were performed for the exhaust plume evolution from an idling aircraft, and we found that the model-predicted volatile PM composition on soot is comparable with those results from past field measurements.
On the Design and Matching of Turbocharger Single Scroll Turbines for Pass Car Gasoline Engines
Authors: Gugau Marc , Roclawski Harald
Published: May 2014

With emission legislation becoming more stringent within the next years, almost all future internal combustion gasoline engines need to reduce specific fuel consumption, most of them by using turbochargers. Additionally, car manufactures attach high importance to a good drivability, which usually is being quantified as a target torque already available at low engine speeds—reached in transient response operation as fast as possible. These engine requirements result in a challenging turbocharger compressor and turbine design task, since for both not one single operating point needs to be aerodynamically optimized but the components have to provide for the optimum overall compromise for maximum thermodynamic performance. The component design targets are closely related and actually controlled by the matching procedure that fits turbine and compressor to the engine. Inaccuracies in matching a turbine to the engine full load are largely due to the pulsating engine flow characteristic and arise from the necessity of arbitrary turbine map extrapolation toward low turbine blade speed ratios and the deficient estimation of turbine efficiency for low engine speed operating points. This paper addresses the above described standard problems, presenting a methodology that covers almost all aspects of thermodynamic turbine design based on a comparison of radial and mixed-flow turbines. Wheel geometry definition with respect to contrary design objectives is done using computational fluid dynamics (CFD), finite element analysis (FEA), and optimization software. Parametrical turbine models, composed of wheel, volute, and standard piping allow for fast map calculation similar to steady hot gas tests but covering the complete range of engine pulsating mass flow. These extended turbine maps are then used for a particular assessment of turbine power output under unsteady flow admission resulting in an improved steady-state matching quality. Additionally, the effect of various design parameters like either volute sizing or the choice of compressor to turbine diameter ratio on turbine blade speed ratio operating range as well as well as turbine inertia effect is analyzed. Finally, this method enables the designer to comparatively evaluate the ability of a turbine design to accelerate the turbocharger speed for transient engine response while still offering a map characteristic that keeps fuel consumption low at all engine speeds.
Use of Yeast Spores for Microencapsulation of Enzymes
Authors: Shi Libing , Li Zijie , Tachikawa Hiroyuki , Gao Xiao-Dong , Nakanishi Hideki
Published: May 2014

Here, we report a novel method to produce microencapsulated enzymes using
Saccharomyces cerevisiae
spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose. This suggests that substrate selectivity could be altered by the encapsulation.
Multilocus Sequence Typing Confirms Wild Birds as the Source of a Campylobacter Outbreak Associated with the Consumption of Raw Peas
Authors: Kwan Patrick S. L. , Xavier Catherine , Santovenia Monica , Pruckler Janet , Stroika Steven , Joyce Kevin , Gardner Tracie , Fields Patricia I. , McLaughlin Joe , Tauxe Robert V. , Fitzgerald Collette
Published: May 2014

From August to September 2008, the Centers for Disease Control and Prevention (CDC) assisted the Alaska Division of Public Health with an outbreak investigation of campylobacteriosis occurring among the residents of Southcentral Alaska. During the investigation, pulsed-field gel electrophoresis (PFGE) of
Campylobacter jejuni
isolates from human, raw pea, and wild bird fecal samples confirmed the epidemiologic link between illness and the consumption of raw peas contaminated by sandhill cranes for 15 of 43 epidemiologically linked human isolates. However, an association between the remaining epidemiologically linked human infections and the pea and wild bird isolates was not established. To better understand the molecular epidemiology of the outbreak,
C. jejuni
isolates (
n
= 130; 59 from humans, 40 from peas, and 31 from wild birds) were further characterized by multilocus sequence typing (MLST). Here we present the molecular evidence to demonstrate the association of many more human
C. jejuni
infections associated with the outbreak with raw peas and wild bird feces. Among all sequence types (STs) identified, 26 of 39 (67%) were novel and exclusive to the outbreak. Five clusters of overlapping STs (
n
= 32 isolates; 17 from humans, 2 from peas, and 13 from wild birds) were identified. In particular, cluster E (
n
= 7 isolates; ST-5049) consisted of isolates from humans, peas, and wild birds. Novel STs clustered closely with isolates typically associated with wild birds and the environment but distinct from lineages commonly seen in human infections. Novel STs and alleles recovered from human outbreak isolates allowed additional infections caused by these rare genotypes to be attributed to the contaminated raw peas.
Gene Discovery for Enzymes Involved in Limonene Modification or Utilization by the Mountain Pine Beetle-Associated Pathogen Grosmannia clavigera
Authors: Wang Ye , Lim Lynette , Madilao Lina , Lah Ljerka , Bohlmann Joerg , Breuil Colette
Published: May 2014

To successfully colonize and eventually kill pine trees,
Grosmannia clavigera
(Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (
Dendroctonus ponderosae
), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone,
p
-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.
Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii
Authors: Ji Xiaofei , Wang Ying , Zhang Cong , Bai Xinfeng , Zhang Weican , Lu Xuemei
Published: May 2014

Cytophaga hutchinsonii
is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of
C. hutchinsonii
. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by
C. hutchinsonii
. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by
C. hutchinsonii
.
Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments
Authors: Denes Thomas , Vongkamjan Kitiya , Ackermann Hans-Wolfgang , Moreno Switt Andrea I. , Wiedmann Martin , den Bakker Henk C.
Published: May 2014

The genus
Listeria
is ubiquitous in the environment and includes the globally important food-borne pathogen
Listeria monocytogenes
. While the genomic diversity of
Listeria
has been well studied, considerably less is known about the genomic and morphological diversity of
Listeria
bacteriophages. In this study, we sequenced and analyzed the genomes of 14
Listeria
phages isolated mostly from New York dairy farm environments as well as one related
Enterococcus faecalis
phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These
Listeria
phages, based on gene orthology and morphology, together with previously sequenced
Listeria
phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large-genome (∼135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (∼66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of
E. faecalis
phages, with medium-sized genomes (∼56 kb), was identified, which grouped together and shares morphological features with the novel
Listeria
phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.
Environmental Surveillance of Human Enteroviruses in Shandong Province, China, 2008 to 2012: Serotypes, Temporal Fluctuation, and Molecular Epidemiology
Authors: Wang Haiyan , Tao Zexin , Li Yan , Lin Xiaojuan , Yoshida Hiromu , Song Lizhi , Zhang Yong , Wang Suting , Cui Ning , Xu Wenbo , Song Yanyan , Xu Aiqiang
Published: May 2014

Environmental surveillance is an effective approach in investigating the circulation of polioviruses (PVs) and other human enteroviruses (EVs) in the population. The present report describes the results of environmental surveillance conducted in Shandong Province, China, from 2008 to 2012. A total of 129 sewage samples were collected, and 168 PVs and 1,007 nonpolio enteroviruses (NPEVs) were isolated. VP1 sequencing and typing were performed on all isolates. All PV strains were Sabin-like, with the numbers of VP1 substitutions ranging from 0 to 7. The NPEVs belonged to 19 serotypes, and echovirus 6 (E6), E11, coxsackievirus B3 (CVB3), E3, E12, and E7 were the six main serotypes, which accounted for 18.3%, 14.8%, 14.5%, 12.9%, 9.0%, and 5.7% of NPEVs isolated, respectively. Typical summer-fall peaks of NPEV were observed in the monthly distribution of isolation, and an epidemic pattern of annual circulation was revealed for the common serotypes. Phylogenetic analysis was performed on environmental CVB3 and E3 strains with global reference strains and local strains from aseptic meningitis patients. Shandong strains formed distinct clusters, and a close relationship was observed between local environmental and clinical strains. As an EV-specific case surveillance system is absent in China and many other countries, continuous environmental surveillance should be encouraged to investigate the temporal circulation and phylogeny of EVs in the population.
Using Signature Genes as Tools To Assess Environmental Viral Ecology and Diversity
Authors: Adriaenssens Evelien M. , Cowan Don A.
Published: May 2014

Viruses (including bacteriophages) are the most abundant biological entities on the planet. As such, they are thought to have a major impact on all aspects of microbial community structure and function. Despite this critical role in ecosystem processes, the study of virus/phage diversity has lagged far behind parallel studies of the bacterial and eukaryotic kingdoms, largely due to the absence of any universal phylogenetic marker. Here we review the development and use of signature genes to investigate viral diversity, as a viable strategy for data sets of specific virus groups. Genes that have been used include those encoding structural proteins, such as portal protein, major capsid protein, and tail sheath protein, auxiliary metabolism genes, such as
psbA
,
psbB
, and
phoH
, and several polymerase genes. These marker genes have been used in combination with PCR-based fingerprinting and/or sequencing strategies to investigate spatial, temporal, and seasonal variations and diversity in a wide range of habitats.
Influence of Hand Rearing and Bird Age on the Fecal Microbiota of the Critically Endangered Kakapo
Authors: Waite David W. , Eason Daryl K. , Taylor Michael W.
Published: May 2014

The critically endangered New Zealand parrot, the kakapo, is subject to an intensive management regime aiming to maintain bird health and boost population size. Newly hatched kakapo chicks are subjected to human intervention and are frequently placed in captivity throughout their formative months. Hand rearing greatly reduces mortality among juveniles, but the potential long-term impact on the kakapo gut microbiota is uncertain. To track development of the kakapo gut microbiota, fecal samples from healthy, prefledged juvenile kakapos, as well as from unrelated adults, were analyzed by using 16S rRNA gene amplicon pyrosequencing. Following the original sampling, juvenile kakapos underwent a period of captivity, so further sampling during and after captivity aimed to elucidate the impact of captivity on the juvenile gut microbiota. Variation in the fecal microbiota over a year was also investigated, with resampling of the original juvenile population. Amplicon pyrosequencing revealed a juvenile fecal microbiota enriched with particular lactic acid bacteria compared to the microbiota of adults, although the overall community structure did not differ significantly among kakapos of different ages. The abundance of key operational taxonomic units (OTUs) was correlated with antibiotic treatment and captivity, although the importance of these factors could not be proven unequivocally within the bounds of this study. Finally, the microbial community structure of juvenile and adult kakapos changed over time, reinforcing the need for continual monitoring of the microbiota as part of regular health screening.
The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions
Authors: Adler Philipp , Frey Lasse Jannis , Berger Antje , Bolten Christoph Josef , Hansen Carl Erik , Wittmann Christoph
Published: May 2014

Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel13C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present.
Novel Microarray Design for Molecular Serotyping of Shiga Toxin-Producing Escherichia coli Strains Isolated from Fresh Produce
Authors: Lacher David W. , Gangiredla Jayanthi , Jackson Scott A. , Elkins Christopher A. , Feng Peter C. H.
Published: May 2014

Serotyping
Escherichia coli
is a cumbersome and complex procedure due to the existence of large numbers of O- and H-antigen types. It can also be unreliable, as many Shiga toxin-producing
E. coli
(STEC) strains isolated from fresh produce cannot be typed by serology or have only partial serotypes. The FDA
E. coli
identification (FDA-ECID) microarray, designed for characterizing pathogenic
E. coli
, contains a molecular serotyping component, which was evaluated here for its efficacy. Analysis of a panel of 75 reference
E. coli
strains showed that the array correctly identified the O and H types in 97% and 98% of the strains, respectively. Comparative analysis of 73 produce STEC strains showed that serology and the array identified 37% and 50% of the O types, respectively, and that the array was able to identify 16 strains that could not be O serotyped. Furthermore, the array identified the H types of 97% of the produce STEC strains compared to 65% by serology, including six strains that were mistyped by serology. These results show that the array is an effective alternative to serology in serotyping environmental
E. coli
isolates.
A Function of SmeDEF, the Major Quinolone Resistance Determinant of Stenotrophomonas maltophilia, Is the Colonization of Plant Roots
Authors: García-León Guillermo , Hernández Alvaro , Hernando-Amado Sara , Alavi Peyman , Berg Gabriele , Martínez José Luis
Published: May 2014

Quinolones are synthetic antibiotics, and the main cause of resistance to these antimicrobials is mutation of the genes encoding their targets. However, in contrast to the case for other organisms, such mutations have not been found in quinolone-resistant
Stenotrophomonas maltophilia
isolates, in which overproduction of the SmeDEF efflux pump is a major cause of quinolone resistance. SmeDEF is chromosomally encoded and highly conserved in all studied
S. maltophilia
strains; it is an ancient element that evolved over millions of years in this species. It thus seems unlikely that its main function would be resistance to quinolones, a family of synthetic antibiotics not present in natural environments until the last few decades. Expression of SmeDEF is tightly controlled by the transcriptional repressor SmeT. Our work shows that plant-produced flavonoids can bind to SmeT, releasing it from
smeDEF
and
smeT
operators. Antibiotics extruded by SmeDEF do not impede the binding of SmeT to DNA. The fact that plant-produced flavonoids specifically induce
smeDEF
expression indicates that they are
bona fide
effectors regulating expression of this resistance determinant. Expression of efflux pumps is usually downregulated unless their activity is needed. Since
smeDEF
expression is triggered by plant-produced flavonoids, we reasoned that this efflux pump may have a role in the colonization of plants by
S. maltophilia
. Our results showed that, indeed, deletion of
smeE
impairs
S. maltophilia
colonization of plant roots. Altogether, our results indicate that quinolone resistance is a recent function of SmeDEF and that colonization of plant roots is likely one original function of this efflux pump.
Cellulolytic Streptomyces Strains Associated with Herbivorous Insects Share a Phylogenetically Linked Capacity To Degrade Lignocellulose
Authors: Book Adam J. , Lewin Gina R. , McDonald Bradon R. , Takasuka Taichi E. , Doering Drew T. , Adams Aaron S. , Blodgett Joshua A. V. , Clardy Jon , Raffa Kenneth F. , Fox Brian G. , Currie Cameron R.
Published: May 2014

Actinobacteria in the genus
Streptomyces
are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of
Streptomyces
to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of
Streptomyces
isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp,
Sirex noctilio
. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely related and that they share similar compositions of carbohydrate-active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted by both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated
Streptomyces
strains with high biomass-degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass.
Expression of a Toll Signaling Regulator Serpin in a Mycoinsecticide for Increased Virulence
Authors: Yang Linzhi , Keyhani Nemat O. , Tang Guirong , Tian Chuang , Lu Ruipeng , Wang Xin , Pei Yan , Fan Yanhua
Published: May 2014

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The
Drosophila melanogaster
serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus
Beauveria bassiana
offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD
50
) for the wild-type parent, the LD
50
of
B. bassiana
expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ∼3-fold, and the median lethal time against the greater wax moth (
Galleria mellonella
) was decreased by ∼24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph.
In vitro
and
in vivo
assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous
B. bassiana
-derived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.
High-Precision Fitting Measurements of the Kinetics of Size Changes during Germination of Individual Bacillus Spores
Authors: Liang Jintao , Zhang Pengfei , Setlow Peter , Li Yong-Qing
Published: May 2014

High-precision measurements of size changes of individual bacterial spores based on ellipse fitting to bright-field images recorded with a digital camera were employed to monitor the germination of
Bacillus
spores with a precision of ∼5 nm. To characterize the germination of individual spores, we recorded bright-field and phase-contrast images and found that the timing of changes in their normalized intensities coincided, so the bright-field images can be used to characterize spore size and refractility changes during germination. The major conclusions from this work were as follows. (i) The sizes of germinating
B. cereus
spores were nearly unchanged until
T
release
, the time of the completion of CaDPA (a 1:1 chelate of Ca
2+
and dipicolinic acid [DPA]) release after addition of nutrient germinants. (ii) The minor axis of germinating
B. cereus
spores rapidly increased by ∼50 nm in a few seconds right after
T
release
, while the major axis was slightly decreased or unchanged. Both the minor and major axes remained unchanged for a further 30 to 45 s and then increased by 100 to 200 nm by
T
lys
, the time of completion of cortex lysis. (iii) Individual spores in a population showed significant heterogeneity in the timing of germination events, such as
T
release
and
T
lys
, but also variation in size changes during germination. (iv)
Bacillus subtilis
wild-type spores,
B. subtilis
spores lacking the cortex-lytic enzyme CwlJ, and wild-type
Bacillus megaterium
spores showed similar kinetics of size changes during nutrient germination. The size increases in germinating spores probably result from uptake of water and cortex lysis after completion of CaDPA release.
Atmospheric N Deposition Increases Bacterial Laccase-Like Multicopper Oxidases: Implications for Organic Matter Decay
Authors: Freedman Zachary , Zak Donald R.
Published: May 2014

Anthropogenic release of biologically available nitrogen (N) has increased dramatically over the last 150 years, which can alter the processes controlling carbon (C) storage in terrestrial ecosystems. In a northern hardwood forest ecosystem located in Michigan in the United States, nearly 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This change occurred concomitantly with compositional changes inBasidiomycetefungi and inActinobacteria, as well as the downregulation of fungal lignocelluloytic genes. Recently, laccase-like multicopper oxidases (LMCOs) have been discovered among bacteria which can oxidize β-O-4 linkages in phenolic compounds (e.g., lignin and humic compounds), resulting in the production of dissolved organic carbon (DOC). Here, we examined how nearly 2 decades of experimental N deposition has affected the abundance and composition of saprotrophic bacteria possessing LMCO genes. In our experiment, LMCO genes were more abundant in the forest floor under experimental N deposition whereas the abundances of bacteria and fungi were unchanged. Experimental N deposition also led to less-diverse, significantly altered bacterial and LMCO gene assemblages, with taxa implicated in organic matter decay (i.e.,Actinobacteria,Proteobacteria) accounting for the majority of compositional changes. These results suggest that experimental N deposition favors bacteria in the forest floor that harbor the LMCO gene and represents a plausible mechanism by which anthropogenic N deposition has reduced decomposition, increased soil C storage, and accelerated phenolic DOC production in our field experiment. Our observations suggest that future rates of atmospheric N deposition could fundamentally alter the physiological potential of soil microbial communities.
Response of a Rice Paddy Soil Methanogen to Syntrophic Growth as Revealed by Transcriptional Analyses
Authors: Liu Pengfei , Yang Yanxiang , Lü Zhe , Lu Yahai
Published: May 2014

Members of
Methanocellales
are widespread in paddy field soils and play the key role in methane production. These methanogens feature largely in these organisms' adaptation to low H
2
and syntrophic growth with anaerobic fatty acid oxidizers. The adaptive mechanisms, however, remain unknown. In the present study, we determined the transcripts of 21 genes involved in the key steps of methanogenesis and acetate assimilation of
Methanocella conradii
HZ254, a strain recently isolated from paddy field soil.
M. conradii
was grown in monoculture and syntrophically with
Pelotomaculum thermopropionicum
(a propionate syntroph) or
Syntrophothermus lipocalidus
(a butyrate syntroph). Comparison of the relative transcript abundances showed that three hydrogenase-encoding genes and all methanogenesis-related genes tested were upregulated in cocultures relative to monoculture. The genes encoding formylmethanofuran dehydrogenase (Fwd), heterodisulfide reductase (Hdr), and the membrane-bound energy-converting hydrogenase (Ech) were the most upregulated among the evaluated genes. The expression of the formate dehydrogenase (Fdh)-encoding gene also was significantly upregulated. In contrast, an acetate assimilation gene was downregulated in cocultures. The genes coding for Fwd, Hdr, and the D subunit of F
420
-nonreducing hydrogenase (Mvh) form a large predicted transcription unit; therefore, the Mvh/Hdr/Fwd complex, capable of mediating the electron bifurcation and connecting the first and last steps of methanogenesis, was predicted to be formed in
M. conradii
. We propose that
Methanocella
methanogens cope with low H
2
and syntrophic growth by (i) stabilizing the Mvh/Hdr/Fwd complex and (ii) activating formate-dependent methanogenesis.
Aminopeptidase N1 (EtAPN1), an M1 Metalloprotease of the Apicomplexan Parasite Eimeria tenella, Participates in Parasite Development
Authors: Gras Simon , Byzia Anna , Gilbert Florence B. , McGowan Sheena , Drag Marcin , Silvestre Anne , Niepceron Alisson , Lecaille Fabien , Lalmanach Gilles , Brossier Fabien
Published: May 2014

Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including
Plasmodium
,
Toxoplasma
,
Cryptosporidium
, and
Eimeria
. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus
Eimeria
, we identified and characterized
Eimeria tenella
aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from
Plasmodium falciparum
(PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs.
Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog
Authors: Freire Eden R. , Malvezzi Amaranta M. , Vashisht Ajay A. , Zuberek Joanna , Saada Edwin A. , Langousis Gerasimos , Nascimento Janaína D. F. , Moura Danielle , Darzynkiewicz Edward , Hill Kent , de Melo Neto Osvaldo P. , Wohlschlegel James A. , Sturm Nancy R. , Campbell David A.
Published: May 2014

Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs
in vitro
and is part of a complex
in vivo
that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.
Dynamics of Expression and Maturation of the Type III Secretion System of Enteropathogenic Escherichia coli
Authors: Yerushalmi Gal , Litvak Yael , Gur-Arie Lihi , Rosenshine Ilan
Published: May 2014

Enteropathogenic
Escherichia coli
(EPEC) is a major cause of food poisoning, leading to significant morbidity and mortality. EPEC virulence is dependent on a type III secretion system (T3SS), a molecular syringe employed by EPEC to inject effector proteins into host cells. The injected effector proteins subvert host cellular functions to the benefit of the infecting bacteria. The T3SS and related genes reside in several operons clustered in the locus of enterocyte effacement (LEE). We carried out simultaneous analysis of the expression dynamics of all the LEE promoters and the rate of maturation of the T3SS. The results showed that expression of the
LEE1
operon is activated immediately upon shifting the culture to inducing conditions, while expression of other LEE promoters is activated only ∼70 min postinduction. Parallel analysis showed that the T3SS becomes functional around 100 min postinduction. The T3SS core proteins EscS, EscT, EscU, and EscR are predicted to be involved in the first step of T3SS assembly and are therefore included among the
LEE1
genes. However, interfering with the temporal regulation of EscS, EscT, EscU, and EscR expression has only a marginal effect on the rate of the T3SS assembly. This study provides a comprehensive description of the transcription dynamics of all the LEE genes and correlates it to that of T3SS biogenesis.
Requirement of the Flagellar Protein Export Apparatus Component FliO for Optimal Expression of Flagellar Genes in Helicobacter pylori
Authors: Tsang Jennifer , Hoover Timothy R.
Published: May 2014

Flagellar biogenesis in
Helicobacter pylori
involves the coordinated expression of flagellar genes with assembly of the flagellum. The
H. pylori
flagellar genes are organized into three regulons based on the sigma factor needed for their transcription (RpoD [σ
80
], RpoN [σ
54
], or FliA [σ
28
]). Transcription of RpoN-dependent genes is activated by a two-component system consisting of the sensor kinase FlgS and the response regulator FlgR. While the cellular cues sensed by the FlgS/FlgR two-component system remain to be elucidated, previous studies revealed that disrupting certain components of the flagellar export apparatus inhibited transcription of the RpoN regulon. FliO is the least conserved of the membrane-bound components of the export apparatus and has not been annotated for any of the
H. pylori
genomes sequenced to date. A PSI-BLAST analysis identified a potential
H. pylori
FliO protein which membrane topology algorithms predict to possess a large N-terminal periplasmic domain that is absent from FliO of
Escherichia coli
and
Salmonella
, the paradigms for flagellar structure/function studies. FliO was necessary for flagellar biogenesis as well as wild-type levels of motility and transcription of RpoN-dependent and FliA-dependent flagellar genes in
H. pylori
strain B128. FliO also appears to be required for wild-type levels of the export apparatus protein FlhA in the membrane. Interestingly, the periplasmic and cytoplasmic domains were somewhat dispensable for flagellar gene regulation and assembly, suggesting that these domains have relatively minor roles in flagellar synthesis.
Intracellular Concentrations of 65 Species of Transcription Factors with Known Regulatory Functions in Escherichia coli
Authors: Ishihama Akira , Kori Ayako , Koshio Etsuko , Yamada Kayoko , Maeda Hiroto , Shimada Tomohiro , Makinoshima Hideki , Iwata Akira , Fujita Nobuyuki
Published: May 2014

The expression pattern of the
Escherichia coli
genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in
E. coli
K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of
E. coli
under various stressful conditions in nature.
Physiological and Proteomic Analysis of Escherichia coli Iron-Limited Chemostat Growth
Authors: Folsom James Patrick , Parker Albert E. , Carlson Ross P.
Published: May 2014

Iron bioavailability is a major limiter of bacterial growth in mammalian host tissue and thus represents an important area of study.
Escherichia coli
K-12 metabolism was studied at four levels of iron limitation in chemostats using physiological and proteomic analyses. The data documented an
E. coli
acclimation gradient where progressively more severe iron scarcity resulted in a larger percentage of substrate carbon being directed into an overflow metabolism accompanied by a decrease in biomass yield on glucose. Acetate was the primary secreted organic by-product for moderate levels of iron limitation, but as stress increased, the metabolism shifted to secrete primarily lactate (∼70% of catabolized glucose carbon). Proteomic analysis reinforced the physiological data and quantified relative increases in glycolysis enzyme abundance and decreases in tricarboxylic acid (TCA) cycle enzyme abundance with increasing iron limitation stress. The combined data indicated that
E. coli
responds to limiting iron by investing the scarce resource in essential enzymes, at the cost of catabolic efficiency (i.e., downregulating high-ATP-yielding pathways containing enzymes with large iron requirements, like the TCA cycle). Acclimation to iron-limited growth was contrasted experimentally with acclimation to glucose-limited growth to identify both general and nutrient-specific acclimation strategies. While the iron-limited cultures maximized biomass yields on iron and increased expression of iron acquisition strategies, the glucose-limited cultures maximized biomass yields on glucose and increased expression of carbon acquisition strategies. This study quantified ecologically competitive acclimations to nutrient limitations, yielding knowledge essential for understanding medically relevant bacterial responses to host and to developing intervention strategies.
Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization
Authors: Boyd Chelsea D. , Smith T. Jarrod , El-Kirat-Chatel Sofiane , Newell Peter D. , Dufrêne Yves F. , O'Toole George A.
Published: May 2014

The localization of the LapA protein to the cell surface is a key step required by
Pseudomonas fluorescens
Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins.