bluuebunny/crossref_metadata_2025 huggingface dataset:
Three independent signalling pathways repress motility in Pseudomonas fluorescens F113
Authors: Navazo Ana , Barahona Emma , Redondo‐Nieto Miguel , Martínez‐Granero Francisco , Rivilla Rafael , Martín Marta
Published: April 2009

Motility is one of the most important traits for rhizosphere colonization by pseudomonads. Despite this importance, motility is severely repressed in the rhizosphere‐colonizing strain Pseudomonas fluorescens F113. This bacterium is unable to swarm under laboratory conditions and produce relatively small swimming haloes. However, phenotypic variants with the ability to swarm and producing swimming haloes up to 300% larger than the wild‐type strain, arise during rhizosphere colonization. These variants harbour mutations in the genes encoding the GacA/GacS two‐component system and in other genes. In order to identify genes and pathways implicated in motility repression, we have used generalized mutagenesis with transposons. Analysis of the mutants has shown that besides the Gac system, the Wsp system and the sadB gene, which have been previously implicated in cyclic di‐GMP turnover, are implicated in motility repression: mutants in the gacS, sadB or wspR genes can swarm and produce swimming haloes larger than the wild‐type strain. Epistasis analysis has shown that the pathways defined by each of these genes are independent, because double and triple mutants show an additive phenotype. Furthermore, GacS, SadB and WspR act at different levels. Expression of the fleQ gene, encoding the master regulator of flagella synthesis is higher in the gacS‐ and sadB‐ backgrounds than in the wild‐type strain and this differential expression is reflected by a higher secretion of the flagellin protein FliC. Conversely, no differences in fleQ expression or FliC secretion were observed between the wild‐type strain and the wspR‐ mutant.
The Beijing Eye Study
Authors: Jonas Jost B. , Xu Liang , Wang Ya Xing
Published: April 2009

Purpose:  This review presents and summarizes the findings of the Beijing Eye Study.

Methods:  The Beijing Eye Study is a population‐based study which included 4439 of 5324 subjects (aged ≥ 40 years) who were initially examined in 2001. The study was repeated in 2006, when 3251 (73.2% of 4439, or 61.1% of 5324) of the original subjects participated. Participants underwent a series of examinations including: refractometry; pneumotonometry; biomicroscopy assisted by slit‐lamp; optical coherence tomography of the anterior segment; photography of the cornea, lens, optic disc, macula and fundus; blood sampling for laboratory tests; blood pressure measurements, and determinations of anthropomorphic parameters. They were also asked to complete a questionnaire which included questions on socioeconomic parameters, and awareness and treatment of ocular and general diseases.

Results:  We present normative data for refractive error, anterior segment measurements, intraocular pressure and optic disc structures and their associations, frequency and causes of visual impairment, blindness and visual field defects, prevalences of trachoma, pterygia, open‐angle glaucoma and angle‐closure glaucoma, cortical, nuclear and posterior subcapsular cataract, age‐related macular degeneration, retinal vein occlusions, diabetes mellitus and diabetic retinopathy, myelinated nerve fibres, and retinitis pigmentosa, and associated and risk factors.

Discussion:  These data may be helpful for dealing with public health issues in China and for assessing associated and risk factors of ocular and general diseases in general.
Manglietia guangzhouensis sp. nov. (Magnoliaceae) from Guangdong, China
Authors: Dong An‐Qiang , Zeng Qing‐Wen , Fu Lin , Xing Fu‐Wu
Published: April 2009

Manglietia guangzhouensis A. Q. Dong, Q. W. Zeng & F. W. Xing, a new species of Magnoliaceae from Guangdong province, China, is described and illustrated. It is closely related to M. pachyphylla H. T. Chang, but differs primarily in its brown villose lower surface of leaves, brown tomentose and stout brachyblasts (peduncles), absence of pedicels, sparsely brown pubescent outer tepals and more numerous ovules (12–14) in each carpel. It is a very rare species with a highly restricted distribution.
In Search of a Balanced Relationship: China, Latin America, and the United States
Authors: Ratliff William
Published: April 2009

This study focuses on the four stages of Chinese relations with Latin America between 1949 and mid‐2008. Ties during the 1950s were limited but directed toward a broad cross‐section of individual Latin Americans. This was abruptly reversed during the Sino‐Soviet dispute of the 1960s by militant advocacy of guerrilla warfare in Latin America. From the early 1970s until the death of Mao Zedong, militant Maoism was blended with a renewed opening of relations, now to military and civilian Latin governments. The final period began with Deng Xiaoping and his reforms and continues to today. This study focuses on Sino‐Latin political and economic relations in general and links to the radical governments of Cuba and Venezuela in particular and weighs the impact of this expansion on Sino‐U.S. relations. It concludes with comments on how China's presence may affect political and economic developments in Latin America itself and how to hone productive cooperation among China, the United States, and Latin nations.
Properties, Characteristics, and Combustion Performance of Sasol Fully Synthetic Jet Fuel
Authors: Moses Clifford A. , Roets Petrus N. J.
Published: April 2009

In 1999, as the only inland petroleum refinery in South Africa was reaching capacity, Sasol gained approval of a semisynthetic jet fuel (SSJF) for civil aviation to augment production and meet the growing demand for jet fuel at the airport in Johannesburg. Prior to this, all jet fuel had to be refined from petroleum sources. SSJF consists of up to 50% of an isoparaffinic kerosene produced from coal using Fischer–Tropsch processes. The production of SSJF remains vulnerable to the production capacity of conventional jet fuel, however. To ensure supply, Sasol has proposed producing a fully synthetic jet fuel (FSJF) using synthetic kerosene streams that contain aromatics and satisfy all the property requirements of international specifications for jet fuel. Being fully synthetic, it was necessary to demonstrate that the fuel is “fit-for-purpose” as jet fuel, i.e., behaves like conventional jet fuel in all aspects of storage and handling as well as air worthiness and flight safety. Four sample blends were developed, covering the practical range of production. Extensive tests on chemistry and physical properties and characteristics demonstrated that Sasol FSJF will be typical of conventional jet fuel. As a final demonstration, the engine manufacturers requested a series of engine and combustor tests to evaluate combustion characteristics, emissions, engine durability, and performance. The performance of the synthetic test fuel was typical of conventional jet fuel. This paper identifies the tests and presents the results demonstrating that Sasol fully synthetic jet fuel is fit-for-purpose as jet fuel for civilian aviation. Sasol FSJF is the first fully synthetic jet fuel approved for unrestricted use.
Residual Stress Measurement by Successive Extension of a Slot: The Crack Compliance Method
Authors: Prime Michael B.
Published: April 2009

This article reviews the technical literature on the determination of a residual stress profile by successive extension of a slot and measurement of the resulting strains or displacements. This technique is known variously in the literature as the crack compliance method, the successive cracking method, the slotting method, and a fracture mechanics based approach. The article briefly summarizes the chronological development of this method and then, to facilitate more detailed review, defines the components that make up the method. The theory section of the article first considers forward method solutions including fracture mechanics, finite element, analytical, and body force methods. Then it examines inverse solutions, including incremental inverses and series expansions. Next, the article reviews all experimental applications of the crack compliance method. Aspects reviewed include the specimen geometry and material, the details of making the slot, the deformation measurement, and the theoretical solutions used to solve for stress. Finally, the article makes a brief qualitative comparison between crack compliance and other residual stress measurement methods. In many situations, the crack compliance method offers several advantages over other methods: improved resolution of residual stress variation with depth; the ability to measure both small and very large parts; measurement of stress intensity factor caused by residual stress; measurement of crack closure stresses; increased sensitivity over other material removal methods; and the ability to measure non-crystalline materials. This review article contains 77 references.
Geometry and Strength of General Helicoidal Shells
Authors: Krivoshapko S. N.
Published: April 2009

The geometry of helical surfaces has been well studied. Several available methods of strength analysis of helicoidal shells give one a choice in solving one-dimensional or two-dimensional problems. The basic problems considered in this review article, which contains 181 references, include geometrical research, approximation and bending of helical surfaces, static analysis of helicoidal shells by analytic and numerical methods, the vibrations of pre-twisted cantilevered plates, helical tubular shells, the generation of helical surfaces by mated surface of revolution, and the application of the helicoidal constructions.
Dynamic Analysis of Plates by Boundary Elements
Authors: Providakis Costas P. , Beskos Dimitri E.
Published: April 2009

A review of boundary element methods for the numerical treatment of free and forced vibrations of flexural plates is presented. The integral formulation and the corresponding numerical solution from the direct boundary element method viewpoint are described for elastic or inelastic flexural plates experiencing small deformations. When the material is elastic the formulation can be either in the frequency or the time domain in conjunction with the elastodynamic or the elastostatic fundamental solution of the corresponding flexural plate problem. When use is made of the elastodynamic fundamental solution, the discretization is essentially restricted to the perimeter of the plate, while an interior discretization in addition to the boundary one is needed when the elastostatic fundamental solution is employed in the formulation. However, the great simplicity of the elastostatic fundamental solution leads eventually to more efficient schemes. Besides, through dual reciprocity techniques one can again restrict the discretization to the plate perimeter. Free vibrations are solved by the determinant method when use is made of the elastodynamic fundamental solution, or by generalized eigenvalue analysis when use is made of the elastostatic fundamental solution. Forced vibrations are solved either in the frequency domain in conjunction with Fourier or Laplace transform or the time domain in conjunction with a step-by-step time integration. When the material is inelastic the problem is formulated incrementally in the time domain in conjunction with the elastostatic fundamental solution and the plate response is obtained through step-by-step time integration. Special formulations such as indirect, Green’s function, symmetric, dual and multiple reciprocity, or boundary collocation ones are also reviewed. Effects such as those of corners, viscoelasticity, anisotropy, inhomogeneity, in-plane forces, shear deformation and rotatory inertia, variable thickness, internal supports, elastic foundation and large defections are discussed as well. Representative numerical examples serve to illustrate boundary element methods and demonstrate their advantages over other numerical methods. This review article includes 150 references.
Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1
Authors: Tahiliani Mamta , Koh Kian Peng , Shen Yinghua , Pastor William A. , Bandukwala Hozefa , Brudno Yevgeny , Agarwal Suneet , Iyer Lakshminarayan M. , Liu David R. , Aravind L. , Rao Anjana
Published: April 2009

Methylation Mediation

Methylation of cytosine bases, 5-methylcytosine (5mC), in DNA plays an important regulatory role in mammalian genomes. Methylation patterns are often inherited across generations, but they can also be dynamic, suggesting that active DNA demethylation pathways exist. One such pathway, best characterized in plants, involves the removal of the 5mC base, and its replacement by C, via a DNA repair mechanism.
Kriaucionis and Heintz
(p.
929
, published online 16 April) now show that, as well as 5mC in mammalian genomes, there are also significant amounts of 5-hydroxymethylcytosine (5hmC) in DNA of Purkinje neurons, which have large nuclei with apparently very little heterochromatin.

Tahiliani
et al.

(p. 930, published online 16 April) find that the protein TET1 is capable of converting 5mC into 5hmC both in vitro and in vivo. 5-Hydroxymethylcytosine is also present in embryonic stem cells, and levels of 5hmC and TET1 show correlated variation during cell differentiation.
Pharmacodynamics of Levofloxacin in a Murine Pneumonia Model of
Pseudomonas aeruginosa
Infection: Determination of Epithelial Lining Fluid Targets
Authors: Louie Arnold , Fregeau Christine , Liu Weiguo , Kulawy Robert , Drusano G. L.
Published: April 2009

The dose choice for
Pseudomonas aeruginosa
remains a matter of debate. The actual exposure targets required for multilog killing of organisms at the primary infection site have not been delineated. We studied
Pseudomonas aeruginosa
PAO1 using a murine model of pneumonia. We employed a large mathematical model to fit all the concentration-time data in plasma and epithelial lining fluid (ELF) as well as colony counts in lung simultaneously for all drug doses. Penetration into ELF was calculated to be approximately 77.7%, as indexed to the ratio of the area under the concentration-time curve for ELF (AUC
ELF
) to the AUC
plasma
. We determined the ELF concentration-time profile required to drive a stasis response as well as 1-, 2-, or 3-log
10
(CFU/g) kill. AUC/MIC ratios of 12.4, 31.2, 62.8, and 127.6 were required to drive these bacterial responses. Emergence of resistance was seen only at the two lowest doses (three of five animals at 50 mg/kg [body weight] and one of five animals at 100 mg/kg). The low exposure targets were likely driven by a low mutational frequency to resistance. Bridging to humans was performed using Monte Carlo simulation. With a 750-mg levofloxacin dose, target attainment rates fell below 90% at 4 mg/liter, 1 mg/liter, and 0.5 mg/liter for 1-, 2-, and 3-log kills, respectively. Given the low exposure targets seen with this strain, we conclude that levofloxacin at a 750-mg dose is not adequate for serious
Pseudomonas aeruginosa
pneumonia as a single agent. More isolates need to be studied to make these observations more robust.
In Vitro Activity of Doripenem against
Burkholderia pseudomallei
Authors: Thamlikitkul Visanu , Trakulsomboon Suwanna
Published: April 2009

The MIC
50
and MIC
90
values of doripenem, determined by Etest, for 110 isolates of
Burkholderia pseudomallei
were 0.5 and 0.75 μg/ml, respectively. There were significant correlations between MICs determined by Etest and MICs determined by agar dilution, MICs determined by Etest and inhibition zone size, and MICs determined by agar dilution and inhibition zone size.
Molecular Analysis of the
gyrA
and
gyrB
Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant
Clostridium difficile
Mutants Selected In Vitro
Authors: Spigaglia Patrizia , Barbanti Fabrizio , Louie Thomas , Barbut Frédéric , Mastrantonio Paola
Published: April 2009

Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of
Clostridium difficile
infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the
C. difficile
toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five
C. difficile
parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10
−6
to 6.6 × 10
−5
for MX and from 1.0 × 10
−6
to 2.4 × 10
−5
for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant
C. difficile
clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant
C. difficile
strains also in vivo.
Computer-Aided Identification of Recognized Drugs as
Pseudomonas aeruginosa
Quorum-Sensing Inhibitors
Authors: Yang Liang , Rybtke Morten Theil , Jakobsen Tim Holm , Hentzer Morten , Bjarnsholt Thomas , Givskov Michael , Tolker-Nielsen Tim
Published: April 2009

Attenuation of
Pseudomonas aeruginosa
virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorum-sensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the
P. aeruginosa
quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorum-sensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections.
Soft-Tissue Penetration of Ceftobiprole in Healthy Volunteers Determined by In Vivo Microdialysis
Authors: Barbour April , Schmidt Stephan , Sabarinath Sreedharan Nair , Grant Maria , Seubert Christoph , Skee Donna , Murthy Bindu , Derendorf Hartmut
Published: April 2009

Ceftobiprole is a promising new broad-spectrum cephalosporin with activity against several multidrug-resistant gram-positive and gram-negative species, including methicillin-resistant
Staphylococcus aureus
. In order to make efficacy predications against these resistant bacteria in soft-tissue infections, i.e., skin and skin structure infections, ceftobiprole's ability to reach the site of action should be explored. Therefore, a microdialysis study was conducted in 12 healthy volunteers to determine the penetration of ceftobiprole into skeletal muscle and subcutaneous (s.c.) adipose tissue after a single intravenous dose of 500 mg. Plasma and tissue interstitial space fluid (ISF) drug concentrations were measured for 24 h from the start of the 2-h intravenous infusion. Pharmacokinetic parameters were determined using noncompartmental analysis. The penetration of ceftobiprole into the ISF of tissues was assessed by comparing the ratios between tissue and plasma of the free drug area under the concentration-time curve (
f
AUC). It was found that ceftobiprole distributes into the muscle (
f
AUC
muscle
/
f
AUC
plasma
of 0.69 ± 0.13) and s.c. adipose tissue (
f
AUC
s.c.adipose
/
f
AUC
plasma
of 0.49 ± 0.28). The concentrations in both skeletal muscle and s.c. adipose tissue met the efficacy breakpoint (percentage of the time that free drug concentrations remained above the MIC) for at least 40% of the 8-h dosing interval for organisms with a MIC of 2 mg/liter. Therefore, ceftobiprole qualifies as a potential agent with drug penetration capabilities to treat complicated skin and skin structure infections due to both gram-negative and gram-positive pathogens with MICs equal to or below 2 mg/liter.
In Vitro Chemosensitization ofPlasmodium falciparumto Antimalarials by Verapamil and Probenecid
Authors: Masseno Victor , Muriithi Steven , Nzila Alexis
Published: April 2009

We tested the effect of probenecid and verapamil in chemosensitizingPlasmodium falciparumto 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo.
High Prevalence of
qnr
Genes in Commensal Enterobacteria from Healthy Children in Peru and Bolivia
Authors: Pallecchi Lucia , Riccobono Eleonora , Mantella Antonia , Bartalesi Filippo , Sennati Samanta , Gamboa Herlan , Gotuzzo Eduardo , Bartoloni Alessandro , Rossolini Gian Maria
Published: April 2009

A remarkable prevalence of
qnrB
(54%) and, at a lower level, of
qnrS
(14%) was discovered in pools of commensal enterobacteria from 310 healthy children living in Peru and Bolivia, using a metagenomic approach. Analysis of randomly selected enterobacterial pools revealed that
qnrB
was mainly carried by
Escherichia coli
and
qnrS
by
Klebsiella pneumoniae
. Investigation of 11
qnrB
-positive isolates and 9
qnrS
-positive isolates revealed the presence of plasmid-borne
qnrB19
(
n
= 8),
qnrB2
(
n
= 2),
qnrB10
(
n
= 1), and
qnrS1
(
n
= 9) genes.
Inter- and Intraspecific Variations of Bacterial Communities Associated with Marine Sponges from San Juan Island, Washington
Authors: Lee On On , Wong Yue Him , Qian Pei-Yuan
Published: April 2009

This study attempted to assess whether conspecific or congeneric sponges around San Juan Island, Washington, harbor specific bacterial communities. We used a combination of culture-independent DNA fingerprinting techniques (terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis [DGGE]) and culture-dependent approaches. The results indicated that the bacterial communities in the water column consisted of more diverse bacterial ribotypes than and were drastically different from those associated with the sponges. High levels of similarity in sponge-associated bacterial communities were found only in
Myxilla incrustans
and
Haliclona rufescens
, while the bacterial communities in
Halichondria panicea
varied substantially among sites. Certain terminal restriction fragments or DGGE bands were consistently obtained for different individuals of
M. incrustans
and
H. rufescens
collected from different sites, suggesting that there are stable or even specific associations of certain bacteria in these two sponges. However, no specific bacterial associations were found for
H. panicea
or for any one sponge genus. Sequencing of nine DGGE bands resulted in recovery of seven sequences that best matched the sequences of uncultured
Proteobacteria
. Three of these sequences fell into the sponge-specific sequence clusters previously suggested. An uncultured alphaproteobacterium and a culturable
Bacillus
sp. were found exclusively in all
M. incrustans
sponges, while an uncultured gammaproteobacterium was unique to
H. rufescens
. In contrast, the cultivation approach indicated that sponges contained a large proportion of
Firmicutes
, especially
Bacillus
, and revealed large variations in the culturable bacterial communities associated with congeneric and conspecific sponges. This study revealed sponge species-specific but not genus- or site-specific associations between sponges and bacterial communities and emphasized the importance of using a combination of techniques for studying microbial communities.
Plutonium(V/VI) Reduction by the Metal-Reducing Bacteria
Geobacter metallireducens
GS-15 and
Shewanella oneidensis
MR-1
Authors: Icopini Gary A. , Lack Joe G. , Hersman Larry E. , Neu Mary P. , Boukhalfa Hakim
Published: April 2009

We examined the ability of the metal-reducing bacteria
Geobacter metallireducens
GS-15 and
Shewanella oneidensis
MR-1 to reduce Pu(VI) and Pu(V). Cell suspensions of both bacteria reduced oxidized Pu [a mixture of Pu(VI) and Pu(V)] to Pu(IV). The rate of plutonium reduction was similar to the rate of U(VI) reduction obtained under similar conditions for each bacteria. The rates of Pu(VI) and U(VI) reduction by cell suspensions of
S. oneidensis
were slightly higher than the rates observed with
G. metallireducens
. The reduced form of Pu was characterized as aggregates of nanoparticulates of Pu(IV). Transmission electron microscopy images of the solids obtained from the cultures after the reduction of Pu(VI) and Pu(V) by
S. oneidensis
show that the Pu precipitates have a crystalline structure. The nanoparticulates of Pu(IV) were precipitated on the surface of or within the cell walls of the bacteria. The production of Pu(III) was not observed, which indicates that Pu(IV) was the stable form of reduced Pu under these experimental conditions. Experiments examining the ability of these bacteria to use Pu(VI) as a terminal electron acceptor for growth were inconclusive. A slight increase in cell density was observed for both
G. metallireducens
and
S. oneidensis
when Pu(VI) was provided as the sole electron acceptor; however, Pu(VI) concentrations decreased similarly in both the experimental and control cultures.
Proteomic and Transcriptomic Analyses Reveal Genes Upregulated bycis-Dichloroethene inPolaromonassp. Strain JS666
Authors: Jennings Laura K. , Chartrand Michelle M. G. , Lacrampe-Couloume Georges , Lollar Barbara Sherwood , Spain Jim C. , Gossett James M.
Published: April 2009

Polaromonassp. strain JS666 is the only bacterial isolate capable of usingcis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathioneS-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from −17.4 to −22.4‰. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C═C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.
Role of GerKB in Germination and Outgrowth of
Clostridium perfringens
Spores
Authors: Paredes-Sabja Daniel , Setlow Peter , Sarker Mahfuzur R.
Published: April 2009

Previous work indicated that
Clostridium perfringens gerKA gerKC
spores germinate significantly, suggesting that
gerKB
also has a role in
C. perfringens
spore germination. We now find that (i)
gerKB
was expressed only during sporulation, likely in the forespore; (ii)
gerKB
spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii)
gerKB
spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.
Biofilm Bacterial Community Structure in Streams Affected by Acid Mine Drainage
Authors: Lear Gavin , Niyogi Dev , Harding Jon , Dong Yimin , Lewis Gillian
Published: April 2009

We examined the bacterial communities of epilithic biofilms in 17 streams which represented a gradient ranging from relatively pristine streams to streams highly impacted by acid mine drainage (AMD). A combination of automated ribosomal intergenic spacer analysis with multivariate analysis and ordination provided a sensitive, high-throughput method to monitor the impact of AMD on stream bacterial communities. Significant differences in community structure were detected among neutral to alkaline (pH 6.7 to 8.3), acidic (pH 3.9 to 5.7), and very acidic (pH 2.8 to 3.5) streams. DNA sequence analysis revealed that the acidic streams were generally dominated by bacteria related to the iron-oxidizing genusGallionella, while the organisms in very acidic streams were less diverse and included a high proportion of acidophilic eukaryotes, including taxa related to the algal generaNaviculaandKlebsormidium. Despite the presence of high concentrations of dissolved metals (e.g., Al and Zn) and deposits of iron hydroxide in some of the streams studied, pH was the most important determinant of the observed differences in bacterial community variability. These findings confirm that any restoration activities in such systems must focus on dealing with pH as the first priority.
Detection ofToxoplasma gondiiOocysts in Water Sample Concentrates by Real-Time PCR
Authors: Yang Wenli , Lindquist H. D. Alan , Cama Vitaliano , Schaefer Frank W. , Villegas Eric , Fayer Ronald , Lewis Earl J. , Feng Yaoyu , Xiao Lihua
Published: April 2009

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection ofToxoplasma gondiioocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection ofT. gondiitachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection ofT. gondiioocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.
Characterization of a Unique Class C Acid Phosphatase from
Clostridium perfringens
Authors: Reilly Thomas J. , Chance Deborah L. , Calcutt Michael J. , Tanner John J. , Felts Richard L. , Waller Stephen C. , Henzl Michael T. , Mawhinney Thomas P. , Ganjam Irene K. , Fales William H.
Published: April 2009

Clostridium perfringens
is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of
C. perfringens
among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including
para
-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated

K
m

s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of P
i
/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical
C. perfringens
isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.
Mitogen-Activated Protein Kinase hog1 in the Entomopathogenic FungusBeauveria bassianaRegulates Environmental Stress Responses and Virulence to Insects
Authors: Zhang Yongjun , Zhao Jianhua , Fang Weiguo , Zhang Jianqing , Luo Zhibing , Zhang Mi , Fan Yanhua , Pei Yan
Published: April 2009

Beauveria bassianais an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. However, its insecticide efficacy in the field is often influenced by adverse environmental factors. Thus, understanding the genetic regulatory processes involved in the response to environmental stress would facilitate engineering and production of a more efficient biocontrol agent. Here, a mitogen-activated protein kinase (MAPK)-encoding gene, Bbhog1, was isolated fromB. bassianaand shown to encode a functional homolog of yeastHIGH-OSMOLARITY GLYCEROL 1(HOG1). A Bbhog1null mutation was generated inB. bassianaby targeted gene replacement, and the resulting mutants were more sensitive to hyperosmotic stress, high temperature, and oxidative stress than the wild-type controls. These results demonstrate the conserved function of HOG1 MAPKs in the regulation of abiotic stress responses. Interestingly, ΔBbhog1mutants exhibited greatly reduced pathogenicity, most likely due to a decrease in spore viability, a reduced ability to attach to insect cuticle, and a reduction in appressorium formation. The transcript levels of two hydrophobin-encoding genes,hyd1andhyd2, were dramatically decreased in a ΔBbhog1mutant, suggesting that Bbhog1 may regulate the expression of the gene associated with hydrophobicity or adherence.
Phylogenetic Analysis Indicates Evolutionary Diversity and Environmental Segregation of Marine Podovirus DNA Polymerase Gene Sequences
Authors: Labonté Jessica M. , Reid Karen E. , Suttle Curtis A.
Published: April 2009

The distribution of viral genotypes in the ocean and their evolutionary relatedness remain poorly constrained. This paper presents data on the genetic diversity and evolutionary relationships of 1.2-kb DNA polymerase (
pol
) gene fragments from podoviruses. A newly designed set of PCR primers was used to amplify DNA directly from coastal sediment and water samples collected from inlets adjacent to the Strait of Georgia, British Columbia, Canada, and from the northeastern Gulf of Mexico. Restriction fragment length polymorphism analysis of 160 cloned PCR products revealed 29 distinct operational taxonomic units (OTUs), with OTUs within a site typically being more similar than those among sites. Phylogenetic analysis of the DNA
pol
gene fragments demonstrated high similarity between some environmental sequences and sequences from the marine podoviruses roseophage SIO1 and cyanophage P60, while others were not closely related to sequences from cultured phages. Interrogation of the CAMERA database for sequences from metagenomics data demonstrated that the amplified sequences were representative of the diversity of podovirus
pol
sequences found in marine samples. Our results indicate high genetic diversity within marine podovirus communities within a small geographic region and demonstrate that the diversity of environmental polymerase gene sequences for podoviruses is far more extensive than previously recognized.
First Gene Cassettes of Integrons as Targets in Finding Adaptive Genes in Metagenomes
Authors: Huang Lionel , Cagnon Christine , Caumette Pierre , Duran Robert
Published: April 2009

The first gene cassettes of integrons are involved in the last adaptation response to changing conditions and are also the most expressed. We propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.
Identifying the Source of Unknown Microcystin Genes and Predicting Microcystin Variants by Comparing Genes within Uncultured Cyanobacterial Cells
Authors: Allender Christopher J. , LeCleir Gary R. , Rinta-Kanto Johanna M. , Small Randall L. , Satchwell Michael F. , Boyer Gregory L. , Wilhelm Steven W.
Published: April 2009

While multiple phylogenetic markers have been used in the culture-independent study of microcystin-producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (
mcyA2
and
mcyB1
) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel
mcyA
genotype previously termed
Microcystis
-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.
Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms
Authors: Stieglmeier Michaela , Wirth Reinhard , Kminek Gerhard , Moissl-Eichinger Christine
Published: April 2009

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (
Clostridium
and
Propionibacterium
) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of
Pseudomonas
and
Paenibacillus
.
Comparative Proteomic Analysis of Tolerance and Adaptation of EthanologenicSaccharomyces cerevisiaeto Furfural, a Lignocellulosic Inhibitory Compound
Authors: Lin Feng-Ming , Qiao Bin , Yuan Ying-Jin
Published: April 2009

The molecular mechanism involved in tolerance and adaptation of ethanologenicSaccharomyces cerevisiaeto inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here,18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles ofS. cerevisiaeunder conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD+, and NADH/NAD+were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology.
Distribution and Rate of Microbial Processes in an Ammonia-Loaded Air Filter Biofilm
Authors: Juhler Susanne , Revsbech Niels Peter , Schramm Andreas , Herrmann Martina , Ottosen Lars D. M. , Nielsen Lars Peter
Published: April 2009

The in situ activity and distribution of heterotrophic and nitrifying bacteria and their potential interactions were investigated in a full-scale, two-section, trickling filter designed for biological degradation of volatile organics and NH
3
in ventilation air from pig farms. The filter biofilm was investigated by microsensor analysis, fluorescence in situ hybridization, quantitative PCR, and batch incubation activity measurements. In situ aerobic activity showed a significant decrease through the filter, while the distribution of ammonia-oxidizing bacteria (AOB) was highly skewed toward the filter outlet. Nitrite oxidation was not detected during most of the experimental period, and the AOB activity therefore resulted in NO
2
−
, accumulation, with concentrations often exceeding 100 mM at the filter inlet. The restriction of AOB to the outlet section of the filter was explained by both competition with heterotrophic bacteria for O
2
and inhibition by the protonated form of NO
2
−
, HNO
2
. Product inhibition of AOB growth could explain why this type of filter tends to emit air with a rather constant NH
3
concentration irrespective of variations in inlet concentration and airflow.
Enumeration of
Escherichia coli
Cells on Chicken Carcasses as a Potential Measure of Microbial Process Control in a Random Selection of Slaughter Establishments in the United States
Authors: Altekruse Sean F. , Berrang Mark E. , Marks Harry , Patel Bharat , Shaw William K. , Saini Parmesh , Bennett Patricia A. , Bailey J. Stan
Published: April 2009

To evaluate whether the number of
Escherichia coli
bacteria in carcass rinses from chicken slaughter establishments could be monitored for the purpose of microbial process control, we drew a random sample from 20 of 127 large USDA-inspected operations. In 2005, every 3 months, two sets of 10 carcass rinses, 100 ml each, were collected from establishments, netting 80 sample sets from the rehang and postchill stages.
E. coli
and
Campylobacter
numbers and
Salmonella
prevalence were measured. Mixed-effect models were used to estimate variance of mean log
10
E. coli
cell numbers of 10-carcass rinse sample sets. Relationships between
E. coli
and
Campylobacter
and
Salmonella
were examined. For 10-carcass rinse sets, at both the rehang and postchill stages the mean log
10
E. coli
CFU/ml fit the logistic distribution better than the normal distribution. The rehang overall mean log
10
E. coli
was 3.3 CFU/ml, with a within-sample set standard deviation of 0.6 CFU/ml. The overall postchill mean log
10
E. coli
was 0.8 CFU/ml, with 13 establishments having mean log
10
E. coli
CFU/ml values of less than 1.0 and 7 having mean values of 1.2 or more. At the midpoint separating these establishments, a mean log
10
E. coli
CFU/ml of 1.1, the within-sample set standard deviation was 0.5 CFU/ml, with smaller standard deviations as means increased. Postchill sample sets with mean log
10
E. coli
counts less than or equal to 1.1 CFU/ml had lower overall prevalence of
Salmonella
and mean log
10
Campylobacter
CFU/ml than sample sets with higher means. These findings regarding reductions in
E. coli
numbers provide insight relevant to microbial process control.
Recovery and Detection of
Escherichia coli
O157:H7 in Surface Water, Using Ultrafiltration and Real-Time PCR
Authors: Mull Bonnie , Hill Vincent R.
Published: April 2009

Enterohemorrhagic
Escherichia coli
O157:H7 (EHEC O157:H7) outbreaks have revealed the need for improved analytical techniques for environmental samples. Ultrafiltration (UF) is increasingly recognized as an effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. This study describes the application of hollow-fiber UF as the primary step for concentrating EHEC O157:H7 seeded into 40-liter samples of surface water, followed by an established culture/immunomagnetic-separation (IMS) method and a suite of real-time PCR assays. Three TaqMan assays were used to detect the
stx1, stx2
, and
rfbE
gene targets. The results from this study indicate that approximately 50 EHEC O157:H7 cells can be consistently recovered from a 40-liter surface water sample and detected by culture and real-time PCR. Centrifugation was investigated and shown to be a viable alternative to membrane filtration in the secondary culture/IMS step when water quality limits the volume of water that can be processed by a filter. Using multiple PCR assay sets to detect
rfbE, stx1
, and
stx2
genes allowed for specific detection of EHEC O157:H7 from strains that do not possess all three genes. The reported sample collection and analysis procedure should be a sensitive and effective tool for detecting EHEC O157:H7 in response to outbreaks of disease associated with contaminated water.
Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System
Authors: Jang Su A , Sung Bong Hyun , Cho Ju Hyun , Kim Sun Chang
Published: April 2009

We describe a novel prokaryotic expression system for the production of cationic antimicrobial peptides (AMPs). The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human gamma interferon) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the coexpressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, ∼100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from 1 liter of
Escherichia coli
culture. Our expression system may represent a universal cost-effective solution for the mass production of intact AMPs in their natural forms.