bluuebunny/crossref_metadata_2025 huggingface dataset:
Cylindrical magnets and ideal solenoids
Authors: Derby Norman , Olbert Stanislaw
Published: February 2010

Both wire-wound solenoids and cylindrical magnets can be approximated as ideal azimuthally symmetric solenoids. We present an exact solution for the magnetic field of an ideal solenoid in an easy to use form. The field is expressed in terms of a single function that can be rapidly computed by means of a compact efficient algorithm, which can be coded as an add-in function to a spreadsheet, making field calculations accessible to introductory students. These expressions are not only accurate but are also as fast as most approximate expressions. We demonstrate their utility by simulating the dropping of a cylindrical magnet through a nonmagnetic conducting tube and comparing the calculation with data obtained from experiments suitable for an undergraduate laboratory.
Nonlinear dynamics of a ball rolling on a surface
Authors: Virgin L. N. , Lyman T. C. , Davis R. B.
Published: February 2010

An underlying potential energy function can provide visual and intuitive insight into a system’s stability and overall behavior. In particular, the motion of a ball moving along a curve or surface in a gravitational field provides a macroscale demonstration of interesting dynamics. We investigate the motion of a small ball rolling along a smooth two-dimensional potential surface. A direct experimental realization of this situation is suitable for demonstrating some classic features of nonlinear dynamics. The results of numerical simulations are directly compared with experimental data. To better characterize the dynamical behavior of the ball, especially when it is undergoing chaotic motion, several descriptive measures are discussed, including time-lag embedding, initial condition maps, power spectra, Lyapunov exponents, and fractal dimensions.
Application of exterior calculus to waveguides
Authors: Ferraro Rafael
Published: February 2010

Exterior calculus is a powerful tool for finding solutions to the electromagnetic field equations. Its strength can be better appreciated when applied to nontrivial configurations. We show how to exploit this tool to obtain the TM and TE modes in hollow cylindrical waveguides. The use of exterior calculus and Lorentz boosts leads straightforwardly to the solutions and their respective power transmitted along the waveguide.
Optical Faraday rotation
Authors: Berman P. R.
Published: February 2010

Three calculations of optical Faraday rotation are presented in which a linearly polarized field is incident on a medium of harmonic oscillators in the presence of a longitudinal magnetic field. The rotation of the plane of polarization of the field is evaluated using classical oscillators and the Lorentz force equation, quantum oscillators and the Heisenberg equations of motion, and quantum oscillators and a Schrödinger equation approach. It is shown that a simple argument, based on the assumption that a circularly polarized field drives either Δm=1 or Δm=−1 transitions on absorption (m is the magnetic quantum number), leads to an incorrect result for the Verdet constant.
Solution of Laplace’s equation for the confining end potentials of a coaxial Malmberg–Penning trap
Authors: Eggleston D. L. , Schroeter Darrell F.
Published: February 2010

Laplace’s equation is solved for the confining potentials at the ends of a coaxial form of the Malmberg–Penning charged particle trap. The solution gives insight into the confinement and dynamics of the trapped particles. The solution employs several mathematical methods that are often studied in isolation. The connections between these methods are illustrated by solving the problem in different ways. This process also produces several new integral and series identities.
Electronic structure, Raman tensors, and resonance phenomena in a simple molecular model
Authors: Meyer M. , Etchegoin P. G. , Le Ru E. C.
Published: February 2010

Some concepts in modern spectroscopy are very specialized, and explanations based on simple examples are not readily available. An example is the changes in the intrinsic symmetry of Raman tensors in molecules produced by resonance or near-resonance conditions. Many of these effects can be obtained from commercial and open source programs that solve the electronic structure of the molecules with density functional theory and compute the Raman tensors of the vibrations. The origin of these changes is hidden by the complexity of these calculations and by the many intermediate computational steps that are not presented to the user. We discuss a simplified model for the electronic structure of a molecule and correlate what is observed with a calculation using density functional theory for a specific molecule. The model yields insight into resonance phenomena, symmetry-related aspects of Raman tensors, and the microscopic origin of the Raman effect itself.
The profile of a capillary liquid bridge between solid surfaces
Authors: van Honschoten J. W. , Tas N. R. , Elwenspoek M.
Published: February 2010

Scanning force microscopy, such as atomic force microscopy (AFM) is complicated by the capillary force of a water meniscus formed in air between the probe tip and the sample. This small liquid bridge between the hydrophilic sample and the sharp AFM tip can be formed by capillary condensation from the vapor phase. We present an analytical model that describes the shape of the meniscus in which the pressure difference across the curved liquid air interface is taken into account. The analysis is based on a minimization of the liquid surface energy, together with the boundary condition of a given pressure drop across the surface as determined by the relative humidity of the vapor. The capillary forces that the wetting liquid exerts on the AFM tip are derived from the model. The resulting expressions can be used to describe arbitrary axial symmetric liquid air interfaces with nonzero total curvature, such as fluid bridges between two surfaces and droplets under a uniform force. The model illustrates some of the basic concepts of capillarity, such as surface tension forces and interfacial pressure drops.
The force, power, and energy of the 100 meter sprint
Authors: Helene O. , Yamashita M. T.
Published: February 2010

At the 2008 Summer Olympics in Beijing, Usain Bolt broke the world record for the 100 m sprint. Just one year later, at the 2009 World Championships in Athletics in Berlin he broke it again. A few months after Beijing, Eriksen et al. [Am. J. Phys. 77, 224–228 (2009)] studied Bolt’s performance and predicted that Bolt could have run about one-tenth of a second faster, which was confirmed in Berlin. In this paper we extend the analysis of Eriksen et al. to model Bolt’s velocity time dependence for the Beijing 2008 and Berlin 2009 records. We deduce the maximum force, the maximum power, and the total mechanical energy produced by Bolt in both races. Surprisingly, we conclude that all of these values were smaller in 2009 than in 2008.
Thermal fluctuations near a phase transition probed through the electrical resistivity of high-temperature superconductors
Authors: Cotón Noelia , Guzmán Francisco J. , Ramallo Manuel V. , Ríos Alexandre , Torrón Carolina , Vidal Félix
Published: February 2010

We present a simple and inexpensive experimental setup that can determine the thermal fluctuations near a superconducting transition. By using equipment common in undergraduate laboratories, the in-plane dc electrical resistivity of a cuprate superconductor as a function of temperature is measured with resolution sufficient to analyze the fluctuation conductivity above the superconducting critical temperature, including the values of the critical exponents. We also present a simple calculation of the fluctuation conductivity, including its dependence on the layered structure of the material, within the Gaussian–Ginzburg–Landau formalism.
Molecular Epidemiology ofCampylobacter jejuniin a Geographically Isolated Country with a Uniquely Structured Poultry Industry
Authors: Müllner Petra , Collins-Emerson Julie M. , Midwinter Anne C. , Carter Philip , Spencer Simon E. F. , van der Logt Peter , Hathaway Steve , French Nigel P.
Published: February 2010

In New Zealand the number of campylobacteriosis notifications increased markedly between 2000 and 2007. Notably, this country's poultry supply is different than that of many developed countries as the fresh and frozen poultry available at retail are exclusively of domestic origin. To examine the possible link between human cases and poultry, a sentinel surveillance site was established to study the molecular epidemiology ofCampylobacter jejuniover a 3-year period from 2005 to 2008 using multilocus sequence typing. Studies showed that 60.1 to 81.4% of retail poultry carcasses from the major suppliers were contaminated withC. jejuni. Differences were detected in the probability and level of contamination and the relative frequency of genotypes for individual poultry suppliers and humans. Some carcasses were contaminated with isolates belonging to more than one sequence type (ST), and there was evidence of both ubiquitous and supplier-associated strains, an epidemiological pattern not recognized yet in other countries. The common poultry STs were also common in human clinical cases, providing evidence that poultry is a major contributor to human infection. Both internationally rare genotypes, such as ST-3069 and ST-474, and common genotypes, such as ST-45 and ST-48, were identified in this study. The dominant human sequence type in New Zealand, ST-474, was found almost exclusively in isolates from one poultry supplier, which provided evidence thatC. jejunihas a distinctive molecular epidemiology in this country. These results may be due in part to New Zealand's geographical isolation and its uniquely structured poultry industry.
Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants
Authors: Nettmann E. , Bergmann I. , Pramschüfer S. , Mundt K. , Plogsties V. , Herrmann C. , Klocke M.
Published: February 2010

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescencein situhybridization (FISH) was carried out to quantify the methanogenicArchaeain the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophicMethanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order,Methanoculleuswas found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic familyMethanosaetaceaewas determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence ofMethanosaetaceaein the biogas reactors and high concentrations of total ammonia (sum of NH3and NH4+) was observed.
Stable Isotope Probing Analysis of Interactions between Ammonia Oxidizers
Authors: Tourna Maria , Freitag Thomas E. , Prosser James I.
Published: February 2010

The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a
13
C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three
Nitrosospira
pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with
13
C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant
Nitrosospira
cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.
The Genome Sequence ofPsychrobacter arcticus273-4, a Psychroactive Siberian Permafrost Bacterium, Reveals Mechanisms for Adaptation to Low-Temperature Growth
Authors: Ayala-del-Río Héctor L. , Chain Patrick S. , Grzymski Joseph J. , Ponder Monica A. , Ivanova Natalia , Bergholz Peter W. , Di Bartolo Genevive , Hauser Loren , Land Miriam , Bakermans Corien , Rodrigues Debora , Klappenbach Joel , Zarka Dan , Larimer Frank , Richardson Paul , Murray Alison , Thomashow Michael , Tiedje James M.
Published: February 2010

Psychrobacter arcticusstrain 273-4, which grows at temperatures as low as −10°C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed byP. arcticus273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of theP. arcticusproteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting thatP. arcticusevolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (−10°C to −12°C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at thesein situtemperatures, which allowsP. arcticusto live at subzero temperatures.
New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the
Rhodococcus
Genome
Authors: Sallam Khalid Ibrahim , Tamura Noriko , Imoto Noriko , Tamura Tomohiro
Published: February 2010

We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the
Rhodococcus
genome in a single step. For this, we cotransformed two vectors into
Rhodococcus
by electroporation: pTip-
istAB
-
sacB
regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-
sacB
provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D
3
hydroxylase into the
Rhodococcus
genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.
Effects of Simulated Mars Conditions on the Survival and Growth of
Escherichia coli
and
Serratia liquefaciens
Authors: Berry Bonnie J. , Jenkins David G. , Schuerger Andrew C.
Published: February 2010

Escherichia coli
and
Serratia liquefaciens
, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of
E. coli
and
S. liquefaciens
cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30°C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl
2
, MgSO
4
, NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for
E. coli
and
S. liquefaciens
at 30 or 20°C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO
4
maintained at 20 or 30°C. Growth rates of
E. coli
and
S. liquefaciens
in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative
E. coli
cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and −50°C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m
−2
for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of
E. coli
failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that
E. coli
may be able to survive, but not grow, in surficial soils on Mars.
Regulation of Morphogenesis and Biocontrol Properties inTrichoderma virensby a VELVET Protein, Vel1
Authors: Mukherjee Prasun K. , Kenerley Charles M.
Published: February 2010

Mycoparasitic strains ofTrichodermaare applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides areTrichodermabased, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, inTrichoderma virens, a commercial biocontrol agent. Mutants with mutations invel1were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains ofTrichoderma, since Vel1 is very highly conserved across threeTrichodermaspecies.
Census of the Viral Metagenome within an Activated Sludge Microbial Assemblage
Authors: Parsley Larissa C. , Consuegra Erin J. , Thomas Stephen J. , Bhavsar Jaysheel , Land Andrew M. , Bhuiyan Nadia N. , Mazher Mustafa A. , Waters Robert J. , Wommack K. Eric , Harper Willie F. , Liles Mark R.
Published: February 2010

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.
Distribution of Virulence Factors and Molecular Fingerprinting of
Aeromonas
Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission
Authors: Khajanchi Bijay K. , Fadl Amin A. , Borchardt Mark A. , Berg Richard L. , Horneman Amy J. , Stemper Mary E. , Joseph Sam W. , Moyer Nelson P. , Sha Jian , Chopra Ashok K.
Published: February 2010

A total of 227 isolates of
Aeromonas
obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the
A. hydrophila
group, the
A. caviae-A. media
group, and the
A. veronii-A. sobria
group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined,
Aeromonas
isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three
Aeromonas
species groups. Likewise, the production of quorum-sensing signaling molecules, such as
N
-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the
A. caviae-A. media
and
A. hydrophila
groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three
Aeromonas
groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the
A. caviae-A. media
group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain
Aeromonas
species after transmission from water to humans.
Isolation of Novel Extreme-Tolerant Cyanobacteria from a Rock-Dwelling Microbial Community by Using Exposure to Low Earth Orbit
Authors: Olsson-Francis Karen , de la Torre Rosa , Cockell Charles S.
Published: February 2010

Many cyanobacteria are known to tolerate environmental extremes. Motivated by an interest in selecting cyanobacteria for applications in space, we launched rocks from a limestone cliff in Beer, Devon, United Kingdom, containing an epilithic and endolithic rock-dwelling community of cyanobacteria into low Earth orbit (LEO) at a height of approximately 300 kilometers. The community was exposed for 10 days to isolate cyanobacteria that can survive exposure to the extreme radiation and desiccating conditions associated with space. Culture-independent (16S rRNA) and culture-dependent methods showed that the cyanobacterial community was composed of
Pleurocapsales
,
Oscillatoriales
, and
Chroococcales
. A single cyanobacterium, a previously uncharacterized extremophile, was isolated after exposure to LEO. We were able to isolate the cyanobacterium from the limestone cliff after exposing the rock-dwelling community to desiccation and vacuum (0.7 × 10
−3
kPa) in the laboratory. The ability of the organism to survive the conditions in space may be linked to the formation of dense colonies. These experiments show how extreme environmental conditions, including space, can be used to select for novel microorganisms. Furthermore, it improves our knowledge of environmental tolerances of extremophilic rock-dwelling cyanobacteria.
Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase
Authors: Schwarze Alexander , Kopczak Marta J. , Rögner Matthias , Lenz Oliver
Published: February 2010

The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H
2
sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O
2
-tolerant membrane-bound hydrogenase (MBH) from
Ralstonia eutropha
H16 to the acceptor site of photosystem I (PS I) from
Synechocystis
sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from
R. eutropha
to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH
PsaE
) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H
2
production capacity of the purified MBH
PsaE
fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH
PsaE
with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH
PsaE
-PS I complex formation and its light-driven H
2
production capacity by means of spectroelectrochemistry.
Sequence-Based Analysis of Secondary-Metabolite Biosynthesis in Marine Actinobacteria
Authors: Gontang Erin A. , Gaudêncio Susana P. , Fenical William , Jensen Paul R.
Published: February 2010

A diverse collection of 60 marine-sediment-derived
Actinobacteria
representing 52 operational taxonomic units was screened by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets were employed to specifically target adenylation domains associated with nonribosomal peptide synthetases (NRPSs) and ketosynthase (KS) domains associated with type I modular, iterative, hybrid, and enediyne polyketide synthases (PKSs). In total, two-thirds of the strains yielded a sequence-verified PCR product for at least one of these biosynthetic types. Genes associated with enediyne biosynthesis were detected in only two genera, while 88% of the ketosynthase sequences shared greatest homology with modular PKSs. Positive strains included representatives of families not traditionally associated with secondary-metabolite production, including the
Corynebacteriaceae
,
Gordoniaceae, Intrasporangiaceae
, and
Micrococcaceae
. In four of five cases where phylogenetic analyses of KS sequences revealed close evolutionary relationships to genes associated with experimentally characterized biosynthetic pathways, secondary-metabolite production was accurately predicted. Sequence clustering patterns were used to provide an estimate of PKS pathway diversity and to assess the biosynthetic richness of individual strains. The detection of highly similar KS sequences in distantly related strains provided evidence of horizontal gene transfer, while control experiments designed to amplify KS sequences from
Salinispora arenicola
strain CNS-205, for which a genome sequence is available, led to the detection of 70% of the targeted PKS pathways. The results provide a bioinformatic assessment of secondary-metabolite biosynthetic potential that can be applied in the absence of fully assembled pathways or genome sequences. The rapid identification of strains that possess the greatest potential to produce new secondary metabolites along with those that produce known compounds can be used to improve the process of natural-product discovery by providing a method to prioritize strains for fermentation studies and chemical analysis.
Laccase and Its Role in Production of Extracellular Reactive Oxygen Species during Wood Decay by the Brown Rot Basidiomycete
Postia placenta
Authors: Wei Dongsheng , Houtman Carl J. , Kapich Alexander N. , Hunt Christopher G. , Cullen Daniel , Hammel Kenneth E.
Published: February 2010

Brown rot basidiomycetes initiate wood decay by producing extracellular reactive oxygen species that depolymerize the structural polysaccharides of lignocellulose. Secreted fungal hydroquinones are considered one contributor because they have been shown to reduce Fe
3+
, thus generating perhydroxyl radicals and Fe
2+
, which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe
3+
tightly, making it unreactive with hydroquinones. For hydroquinone-driven hydroxyl radical production to contribute in this environment, an alternative mechanism to oxidize hydroquinones is required. We show here that aspen wood undergoing decay by the oxalate producer
Postia placenta
contained both 2,5-dimethoxyhydroquinone and laccase activity. Mass spectrometric analysis of proteins extracted from the wood identified a putative laccase (Joint Genome Institute
P. placenta
protein identification number 111314), and heterologous expression of the corresponding gene confirmed this assignment. Ultrafiltration experiments with liquid pressed from the biodegrading wood showed that a high-molecular-weight component was required for it to oxidize 2,5-dimethoxyhydroquinone rapidly and that this component was replaceable by
P. placenta
laccase. The purified laccase oxidized 2,5-dimethoxyhydroquinone with a second-order rate constant near 10
4
M
−1
s
−1
, and measurements of the H
2
O
2
produced indicated that approximately one perhydroxyl radical was generated per hydroquinone supplied. Using these values and a previously developed computer model, we estimate that the quantity of reactive oxygen species produced by
P. placenta
laccase in wood is large enough that it likely contributes to incipient decay.
Changes in Denitrifier Abundance, Denitrification Gene mRNA Levels, Nitrous Oxide Emissions, and Denitrification in Anoxic Soil Microcosms Amended with Glucose and Plant Residues
Authors: Henderson Sherri L. , Dandie Catherine E. , Patten Cheryl L. , Zebarth Bernie J. , Burton David L. , Trevors Jack T. , Goyer Claudia
Published: February 2010

In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N2O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h.nosZgene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels ofPseudomonas mandeliiand closely related species (nirSP) increased only in glucose-amended soil: thenirSPguild abundance increased 5-fold over the 72-h incubation period (P< 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P< 0.001) and then subsequently decreased. ThenosZgene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1,nosZgene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 × 107gene copies and 6.9 × 107transcripts g−1dry soil. Cumulative N2O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. ThenirSPandnosZcommunities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios.
Organization of the Electron Transfer Chain to Oxygen in the Obligate Human Pathogen
Neisseria gonorrhoeae
: Roles for Cytochromes
c
4
and
c
5
, but Not Cytochrome
c
2
, in Oxygen Reduction
Authors: Li Ying , Hopper Amanda , Overton Tim , Squire Derrick J. P. , Cole Jeffrey , Tovell Nicholas
Published: February 2010

Although
Neisseria gonorrhoeae
is a prolific source of eight
c
-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes
c
2
,
c
4
, and
c
5
, encoded by the genes
cccA
,
cycA
, and
cycB
, respectively, have been investigated. Single mutations in genes for either cytochrome
c
4
or
c
5
resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible copy of the
cycA
or
cycB
gene. In contrast, a
cccA
mutant reduced oxygen slightly more rapidly than the parent, suggesting that
cccA
is expressed but cytochrome
c
2
is not involved in electron transfer to cytochrome oxidase. The deletion of
cccA
increased the sensitivity of the
cycB
mutant to excess oxygen but decreased the sensitivity of the
cycA
mutant. Despite many attempts, a double mutant defective in both cytochromes
c
4
and
c
5
could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible
cycB
gene with deletions in both
cycA
and
cycB
was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome
c
4
or
c
5
. These results define the gonococcal electron transfer chain to oxygen in which cytochromes
c
4
and
c
5
, but not cytochrome
c
2
, provide alternative pathways for electron transfer from the cytochrome
bc
1
complex to the terminal oxidase cytochrome
cbb
3
.
Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in
Salmonella enterica
Serovar Typhimurium
Authors: Kim Byoungkwan , Richards Susan M. , Gunn John S. , Slauch James M.
Published: February 2010

Salmonella enterica
serovar Typhimurium replicates in macrophages, where it is subjected to antimicrobial substances, including superoxide, antimicrobial peptides, and proteases. The bacterium produces two periplasmic superoxide dismutases, SodCI and SodCII. Although both are expressed during infection, only SodCI contributes to virulence in the mouse by combating phagocytic superoxide. The differential contribution to virulence is at least partially due to inherent differences in the SodCI and SodCII proteins that are independent of enzymatic activity. SodCII is protease sensitive, and like other periplasmic proteins, it is released by osmotic shock. In contrast, SodCI is protease resistant and is retained within the periplasm after osmotic shock, a phenomenon that we term “tethering.” We hypothesize that in the macrophage, antimicrobial peptides transiently disrupt the outer membrane. SodCII is released and/or phagocytic proteases gain access to the periplasm, and SodCII is degraded. SodCI is tethered within the periplasm and is protease resistant, thereby remaining to combat superoxide. Here we test aspects of this model. SodCII was released by the antimicrobial peptide polymyxin B or a mouse macrophage antimicrobial peptide (CRAMP), while SodCI remained tethered within the periplasm. A
Salmonella pmrA
constitutive mutant no longer released SodCII
in vitro
. Moreover, in the constitutive
pmrA
background, SodCII could contribute to survival of
Salmonella
during infection. SodCII also provided a virulence benefit in mice genetically defective in production of CRAMP. Thus, consistent with our model, protecting the outer membrane against antimicrobial peptides allows SodCII to contribute to virulence
in vivo
. These data also suggest direct
in vivo
cooperative interactions between macrophage antimicrobial effectors.
Polymyxin B Resistance in El Tor
Vibrio cholerae
Requires Lipid Acylation Catalyzed by MsbB
Authors: Matson Jyl S. , Yoo Hyun Ju , Hakansson Kristina , DiRita Victor J.
Published: February 2010

Antimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. In
Vibrio cholerae
, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classical
V. cholerae
mutants resistant to polymyxin B and El Tor
V. cholerae
mutants sensitive to polymyxin B. Insertions in a gene annotated
msbB
(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance of
V. cholerae
. Analysis of a defined mutation in the El Tor biotype demonstrated that
msbB
is required for resistance to all antimicrobial peptides tested. Mutation of
msbB
in a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.
msbB
mutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of the
msbB
mutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase in
V. cholerae
.
Cell Surface Xylanases of the Glycoside Hydrolase Family 10 Are Essential for Xylan Utilization byPaenibacillussp. W-61 as Generators of Xylo-Oligosaccharide Inducers for the Xylanase Genes
Authors: Fukuda Mutsumi , Watanabe Seiji , Yoshida Shigeki , Itoh Hiroya , Itoh Yoshifumi , Kamio Yoshiyuki , Kaneko Jun
Published: February 2010

Paenibacillussp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes,xyn1,xyn3, andxyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A Δxyn5mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5ΔSLH) grew poorly, secreting Xyn5ΔSLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intactxyn5produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer forxyn1andxyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer forxyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all thexyngenes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of thexynsystem.
Heterochronic Phosphorelay Gene Expression as a Source of Heterogeneity in
Bacillus subtilis
Spore Formation
Authors: de Jong Imke G. , Veening Jan-Willem , Kuipers Oscar P.
Published: February 2010

In response to limiting nutrient sources and cell density signals,
Bacillus subtilis
can differentiate and form highly resistant endospores. Initiation of spore development is governed by the master regulator Spo0A, which is activated by phosphorylation via a multicomponent phosphorelay. Interestingly, only part of a clonal population will enter this developmental pathway, a phenomenon known as sporulation bistability or sporulation heterogeneity. How sporulation heterogeneity is established is largely unknown. To investigate the origins of sporulation heterogeneity, we constructed promoter-green fluorescent protein (GFP) fusions to the main phosphorelay genes and perturbed their expression levels. Using time-lapse fluorescence microscopy and flow cytometry, we showed that expression of the phosphorelay genes is distributed in a unimodal manner. However, single-cell trajectories revealed that phosphorelay gene expression is highly dynamic or “heterochronic” between individual cells and that stochasticity of phosphorelay gene transcription might be an important regulatory mechanism for sporulation heterogeneity. Furthermore, we showed that artificial induction or depletion of the phosphorelay phosphate flow results in loss of sporulation heterogeneity. Our data suggest that sporulation heterogeneity originates from highly dynamic and variable gene activity of the phosphorelay components, resulting in large cell-to-cell variability with regard to phosphate input into the system. These transcriptional and posttranslational differences in phosphorelay activity appear to be sufficient to generate a heterogeneous sporulation signal without the need of the positive-feedback loop established by the sigma factor SigH.
The Abi Proteins and Their Involvement in Bacteriocin Self-Immunity
Authors: Kjos Morten , Snipen Lars , Salehian Zhian , Nes Ingolf F. , Diep Dzung B.
Published: February 2010

The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g.,
pln
locus from
Lactobacillus plantarum
and
sag
locus from
Streptococcus pyogenes
), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from
Lactobacillus sakei
23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (
skkAB
) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (
skkI
) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene
plnI
and the Abi-like gene
plnL
from
L. plantarum
also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.
Strains of the East Asian (W/Beijing) Lineage ofMycobacterium tuberculosisAre DosS/DosT-DosR Two-Component Regulatory System Natural Mutants
Authors: Fallow Ashley , Domenech Pilar , Reed Michael B.
Published: February 2010

As part of our ongoing efforts to uncover the phenotypic consequences of genetic variability among clinicalMycobacterium tuberculosisisolates, we previously reported that isolates of the “East Asian” or “W/Beijing” lineage constitutively overexpress the coordinately regulated transcriptional program known as the DosR regulon under standardin vitroconditions. This phenotype distinguishes the W/Beijing lineage from all otherM. tuberculosislineages, which normally induce expression of this regulon only once exposed to low oxygen or nitric oxide, both of which result in inhibition of bacterial respiration and replication. Transcription of the DosR regulon is controlled through a two-component regulatory system comprising the transcription factor DosR and two possible cognate histidine sensor kinases, DosS and DosT. Through sequence analysis of a carefully selected set of isolates representing each of the majorM. tuberculosislineages, we describe herein a naturally occurring frameshift mutation in the gene encoding the DosT sensor kinase for isolates of the most recently evolved W/Beijing sublineages. Intriguingly, the occurrence of the frameshift mutation correlates precisely with the appearance of the constitutive DosR regulon phenotype displayed by the same “modern” W/Beijing strains. However, complementation studies have revealed that the mutation indosTalone is not directly responsible for the constitutive DosR regulon phenotype. Our data serve to highlight the evolutionary pressure that exists among distinctM. tuberculosislineages to maintain tight control over DosR regulon expression.