bluuebunny/crossref_metadata_2025 huggingface dataset:
Self-excited Vibration Caused by Internal Friction in Universal Joints and Its Stabilizing Method
Authors: Saigo M. , Okada Y. , Ono K.
Published: February 2008

Stability of a rotor system induced by Coulomb friction in joints is investigated both theoretically and experimentally. The vibrating system consists of two shafts supported flexibly and connected by three universal joints including a cross-groove (CG) joint. The Coulomb friction in a CG joint, which is one of the most destabilizing joints among practically used ones, is treated. The friction is assumed to be a function of the relative angular velocity between the connected shafts. Three stabilizing effects are investigated: the initial intersecting angle in the CG joint, the asymmetry of the support stiffness and a dynamic damper (D/D). As a result, it is found that the initial intersecting angle has a remarkable stabilizing effect; D/D expands the stable region of the system with an initial intersecting angle; the asymmetry of the support stiffness also has a stabilizing effect on the system with an initial intersecting angle. These stabilizing effects are experimentally confirmed.
Harmonically Forced Wave Propagation in Elastic Cables With Small Curvature
Authors: Behbahani-Nejad M. , Perkins N. C.
Published: February 2008

This study presents an investigation of coupled longitudinal-transverse waves that propagate along an elastic cable. The coupling considered derives from the equilibrium curvature (sag) of the cable. A mathematical model is presented that describes the three-dimensional nonlinear response of an extended elastic cable. An asymptotic form of this model is derived for the linear response of cables having small equilibrium curvature. Linear, in-plane response is described by coupled longitudinal-transverse partial differential equations of motion, which are comprehensively evaluated herein. The spectral relation governing propagating waves is derived using transform methods. In the spectral relation, three qualitatively distinct regimes exist that are separated by two cut-off frequencies which are strongly influenced by cable curvature. This relation is employed in deriving a Green’s function which is then used to construct solutions for in-plane response under arbitrarily distributed harmonic excitation. Analysis of forced response reveals the existence of two types of periodic waves which propagate through the cable, one characterizing extension-compressive deformations (rod-type) and the other characterizing transverse deformations (string-type). These waves may propagate or attenuate depending on wave frequency. The propagation and attenuation of both wave types are highlighted through solutions for an infinite cable subjected to a concentrated harmonic excitation source.
Effects of Shear Deformation and Rotary Inertia on the Free Vibration of a Rotating Annular Plate
Authors: Coˆte´ A. , Atalla N. , Nicolas J.
Published: February 2008

This Tech Brief addresses the effects of shear deformation and rotary inertia on the free vibration of a rotating annular plate. Several studies have been done to understand these effects (Irie et al, 1982; Sinha, 1987). In particular, the importance of these terms is well known for disks with a thickness ratio h/a > 0.05 where h is the thickness of the disk and a is its outer radius. However, there is a need to show how they affect the disk vibration as the thickness ratio increases. It is well accepted that shear deformation and rotary inertia should be included in the analysis when h/a > 0.05 (Batoz and Dhatt, 1990). However, this Tech Brief will show that this criterion is not valid for all the modes. It will present the evolution of the number of modes correctly evaluated versus the thickness ratio, and, finally, will give a heuristic criterion to predict when it is necessary to consider shear deformation and rotary inertia.
On an Innovative Method of Modeling General Nonlinear Mechanical Systems, Part 1: Theory and Numerical Simulations
Authors: Xu K. Q. , Rice H. J.
Published: February 2008

This is the first part of a paper on an innovative approach of studying general nonlinear structural dynamic systems. In this part, the theoretical background and some numerical simulations are presented. Firstly, a frequency domain nonlinear compensation method is proposed. It can be used to recover the linear path of a general non-linear system as long as the Volterra expansion is held between the input and the output. Secondly, a new nonlinear element location scheme is developed based on the system linear path identification. Lastly, as a natural extension in the development of the contents of this paper, an in-situ nonlinear restoring force state mapping technique is proposed. The use of these new methods is demonstrated through several numerical examples of either single or multiple DOF systems incorporating some typical types of nonlinearities.
Large In-Plane Motion of a Rotor
Authors: Cveticanin L.
Published: February 2008

The vibrations of the in-plane rotor system with nonlinear elastic and geometric properties are considered in this paper. The vibrations of the rotor are described as the system often partial differential equations and ten boundary conditions. Approximate Galerkin procedure is used to obtain the second order nonlinear differential equation. The response frequencies of the system are discussed. It is shown that they are functions of angular velocity of the rotor and nonlinear properties of the system.
Free Hydroelastic Vibrations of a Liquid Attached to a Cylindrical Sector Shell in a Zero-Gravity Condition
Authors: Chiba M. , Bauer H. F.
Published: February 2008

A theoretical analysis has been carried out on the linear free hydroelastic vibrations of a liquid attached to a cylindrical sector shell. The liquid is assumed to be an ideal liquid with a free surface, while for the motion of the sector shell the equations of Flu¨gge were employed. The coupled problem has been reduced to an eigenvalue problem by applying the Galerkin method and yields the coupled natural frequencies and vibration modes as eigenvalues and eigenvectors, respectively. Numerical calculations were carried out to find the influence of the parameters of the structure-liquid system.
Active Reduction of Radiated Noise From a Baffled Piston Using a Volume Velocity, Feedforward Controller
Authors: Naghshineh K. , Mason V. B. , Kamman J. W.
Published: February 2008

A new active noise-control technique has been developed for control of low-frequency sound generated by vibrating surfaces which is based on minimizing the volume velocity. Noise reduction is achieved by distributing an array of control devices over the surface of the radiating structure. Each device consists of a motion-sensing mechanism, an analog control circuit, and a loudspeaker. The loudspeaker is driven such that it reduces the volume velocity of the radiating structure within its close proximity. This paper briefly presents the theory behind this approach as well as controller design issues. Finally, a discussion of the experimental verification of this concept using a 10-in. uniformly vibrating circular plate (i.e., a baffled piston) and a single noise-control device is given. Broadband (50–500 Hz) sound reductions in the range of 10–20 dB were achieved over a wide spatial area.
In-Plane Vibration and Crack Detection of a Rotating Shaft-Disk Containing a Transverse Crack
Authors: Wu Ming-Chuan , Huang Shyh-Chin
Published: February 2008

Dynamic response and stability of a rotating shaft-disk containing a transverse crack is investigated. FFT analysis of response amplitudes showed that the 2Ω component (Ω: rotation speed) was excited by crack breathing and could serve as a good index for crack identification. Intensive numerical studies of crack location, crack depth, rotation speed, and sensing position on response amplitudes displayed a feasible technique for the identification of crack depth and crack location. It is achieved by intersecting the two equi-amplitude response curves of two separated sensing probes. Finally, the instability of the system caused by a crack is examined via Floquet theory and the multiple scale method. The stability diagrams, illustrated as functions of crack depth, rotation speed, and damping, are shown and discussed.
Power Flow in Linear, Active Vibration Isolation Systems
Authors: Mahajan S. , Redfield R.
Published: February 2008

Power flow and net energy transfer are examined for actively controlled, vibration isolation dynamics. Fundamental concepts of active damping systems are studied by examining the average power flow in the controlled and passive actuators subject to harmonic inputs. Expressions for energies delivered and absorbed by the actuators per cycle are derived for one and two degree-of-freedom vibration models. Sensitivity to system parameters on power flow in the passive and active components is examined. This analysis tool can be useful in the design of low-power active and regenerative systems.
Vector Triggering Random Decrement for High Identification Accuracy
Authors: Ibrahim S. R. , Asmussen J. C. , Brincker R.
Published: February 2008

Using the Random Decrement (RD) technique to obtain free response estimates and combining this with time domain modal identification methods to obtain the poles and the mode shapes is acknowledged as a fast and accurate way of analysing measured responses of structures subject to ambient loads. When commonly accepted triggering conditions are used however, the user is restricted to use a combination of auto RD and cross RD functions with high noise contents on the cross RD functions. Use of the auto RD functions alone causes the loss of phase information and thus the possibility of estimating mode shapes. In this paper a new algorithm based on pure auto triggering is suggested. Equivalent auto RD functions are estimated for all channels to obtain functions with a minimum of noise, using a vector triggering condition that preserves phase information, and thus, allows for estimation of both poles and mode shapes. The proposed technique (VRD) is compared with the traditional RD technique by evaluating modal parameters extracted from the RD and the VRD functions using ITD identification technique on simulated and experimentally obtained data.
Dynamics of Branched Pipeline Systems Conveying Internal Unsteady Flow
Authors: Lee Usik , Kim Joohong
Published: February 2008

The pipeline system conveying high pressurized unsteady internal flow may experience severe transient vibrations due to the fluid-pipe interaction under the time-varying conditions imposed by the pump and valve operations. In the present work, a set of fully coupled dynamic equations of motion for the pipeline system are developed to include the effect of the circumferential strain due to the internal fluid pressure. A finite element formulation for the fully coupled dynamic equations of motion is introduced and applied to several sample pipeline systems. The connectivity conditions for both fluid and structural variables at the junction of a branched pipeline system are properly incorporated in the finite element formulation. To ensure the validity and accuracy of the present theory of pipedynamics, the same pipeline system considered in a reference work is revisited and the present numerical results are compared with those given in the reference work. A series pipeline system with high reservoir head is then analyzed to investigate the effect of the additional linear/nonlinear coupling terms in the present pipedynamic theory. Numerical tests show that the nonlinear coupling terms may become significant at high fluid pressure and velocity.
On the Influence of Angle-of-Attack or Sideslip on Far-Field Propeller Noise
Authors: Campos L. M. B. C.
Published: February 2008

The effect of angle-of-attack or sideslip on noise radiation by a propeller is considered, (Fig. 3). For a single propeller, neglecting installation effects, the angles of attack and sideslip can be combined (Fig. 2). The simplest model of the propeller, as a point multipole source, leads to an effect of inflow angle on acoustic radiation intensity, which is specified by the Doppler factor (Fig. 1). This simple model gives results consistent with experiments (Zandbergen, Sarin & Donnely, 1984, 1990), with a scaled propeller in a wind tunnel; the simple alberaic formula (Section 2), gives better results than a more elaborate numerical method (Succi, 1979). Proceeding to consider finite source distributions along the blades, leads to a less simple analytical theory (Section 3), based on radiation integrals valid in the far-field. It allows the consideration of unsteady monopole, dipole and quadrupole sources. This analytical theory, allowing for a distribution of acoustic sources along the span of the blades, gives a non-negligible correction to the algebraic theory. Thus the latter is adequate only as a simple order-of-magnitude estimate of noise level; a more acurate calculation noise levels requires evaluation of the radiation integrals, based on a knowledge of the acoustic source distribution.
Targeted Mutagenesis of
Burkholderia thailandensis
and
Burkholderia pseudomallei
through Natural Transformation of PCR Fragments
Authors: Thongdee Metawee , Gallagher Larry A. , Schell Mark , Dharakul Tararaj , Songsivilai Sirirurg , Manoil Colin
Published: March 2008

Burkholderia pseudomallei
is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and
B. thailandensis
is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying
loxP
and
FRT
recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two
Burkholderia
species.
Characterization of an Endo-β-1,6-Galactanase from
Streptomyces avermitilis
NBRC14893
Authors: Ichinose Hitomi , Kotake Toshihisa , Tsumuraya Yoichi , Kaneko Satoshi
Published: March 2008

The putative endo-β-1,6-galactanase gene from
Streptomyces avermitilis
was cloned and expressed in
Escherichia coli
, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote.
Development of Bottom-Fermenting
Saccharomyces
Strains That Produce High SO
2
Levels, Using Integrated Metabolome and Transcriptome Analysis
Authors: Yoshida Satoshi , Imoto Jun , Minato Toshiko , Oouchi Rie , Sugihara Mao , Imai Takeo , Ishiguro Tatsuji , Mizutani Satoru , Tomita Masaru , Soga Tomoyoshi , Yoshimoto Hiroyuki
Published: March 2008

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting
Saccharomyces
strains that produce high levels of SO
2
is desirable, it is complicated by the fact that undesirable H
2
S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO
2
-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that
O
-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO
2
and H
2
S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO
2
, resulting in high SO
2
and low H
2
S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO
2
than the parent but produced parental levels of H
2
S.
Analysis of Bacterial Communities in Soil by Use of Denaturing Gradient Gel Electrophoresis and Clone Libraries, as Influenced by Different Reverse Primers
Authors: Brons Jolanda K. , van Elsas Jan Dirk
Published: March 2008

To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla,
Firmicutes
and
Proteobacteria
. Within
Firmicutes
, the majority of the clones belonged to the genus
Bacillus
. Within
Proteobacteria
, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were
Actinobacteria
,
Acidobacteria
, V
errucomicrobia
,
Chloroflexi
,
Gemmatimonadetes
,
Chlorobi
,
Bacteroidetes
,
Chlamydiae
, candidate division TM7,
Ferribacter
,
Cyanobacteria
, and
Deinococcus
. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.
Saline-Dependent Regulation of Manganese Peroxidase Genes in the Hypersaline-Tolerant White Rot Fungus
Phlebia
sp. Strain MG-60
Authors: Kamei Ichiro , Daikoku Chieko , Tsutsumi Yuji , Kondo Ryuichiro
Published: March 2008

The expression pattern of manganese peroxidases (MnPs) in nitrogen-limited cultures of the saline-tolerant fungus
Phlebia
sp. strain MG-60 is differentially regulated under hypersaline conditions at the mRNA level. When MG-60 was cultured in nitrogen-limited medium (LNM) containing 3% (wt/vol) sea salts (LN-SSM), higher activity of MnPs was observed than that observed in normal medium (LNM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that two MnP isoenzymes were de novo synthesized in the culture of LN-SSM. Three MnP-encoding genes (MG
mnp1
, MG
mnp2
, and MG
mnp3
) were isolated by reverse transcription (RT)-PCR and rapid amplification of cDNA ends PCR techniques. The corresponding isozymes were identified by peptide mass fingerprinting analysis. MnP isozymes encoded by MG
mnp2
and MG
mnp3
were observed mainly in LN-SSM. Real-time RT-PCR analysis revealed high levels of MG
mnp2
and MG
mnp3
transcripts in LN-SSM 48 h after the addition of 2% NaCl. The induction of MnP production and the accumulation of gene transcripts by saline were well correlated in the presence of Mn
2+
. However, in the absence of Mn
2+
, there was no clear correlation between
mnp
transcripts levels and MnP activity, suggesting posttranscriptional regulation by Mn
2+
.
Optical Tweezers Cause Physiological Damage to
Escherichia coli
and
Listeria
Bacteria
Authors: Rasmussen M. B. , Oddershede L. B. , Siegumfeldt H.
Published: March 2008

We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species
Escherichia coli
and three gram-positive species,
Listeria monocytogenes
,
Listeria innocua
, and
Bacillus subtilis
. All cells exhibited some degree of physiological damage, but optically trapped
E. coli
and
L. innocua
cells and a subpopulation of
L. monocytogenes
cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of
Listeria monocytogenes
cells exhibited signs of physiological damage even while trapped at 6 mW, as did
B. subtilis
cells. Increasing the laser power to 18 mW caused the pH gradient of both
Listeria
and
E. coli
cells to decrease within minutes. Moreover, both species of
Listeria
exhibited more-pronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.
Leaf Age as a Risk Factor in Contamination of Lettuce withEscherichia coliO157:H7 andSalmonella enterica
Authors: Brandl M. T. , Amundson R.
Published: March 2008

Outbreaks ofEscherichia coliO157:H7 infections have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of romaine lettuce plants. The increases in population size ofE. coliO157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and the presence of free water on the leaves and varied significantly with leaf age. The population size was consistently ca. 10-fold higher on the young (inner) leaves than on the middle leaves. The growth rates ofSalmonella entericaand of the natural bacterial microflora were similarly leaf age dependent. Both enteric pathogens also achieved higher population sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects preharvest as well as postharvest colonization. Elemental analysis of the exudates collected from the surfaces of leaves of different ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not glucose, to middle leaves enhanced the growth ofE. coliO157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age and nitrogen content contribute to shaping the bacterial communities of preharvest and postharvest lettuce and that young lettuce leaves may be associated with a greater risk of contamination withE. coliO157:H7.
Gene Expression by the Sulfate-Reducing Bacterium
Desulfovibrio vulgaris
Hildenborough Grown on an Iron Electrode under Cathodic Protection Conditions
Authors: Caffrey Sean M. , Park Hyung Soo , Been Jenny , Gordon Paul , Sensen Christoph W. , Voordouw Gerrit
Published: March 2008

The genome sequence of the sulfate-reducing bacterium
Desulfovibrio vulgaris
Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a
c
-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type
D. vulgaris
grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of −1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the
hyn-1
genes for [NiFe] hydrogenase 1 and the
hyd
genes, encoding [Fe] hydrogenase. The
hmc
genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the
hys
genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with
hyn-1
,
hyd
, and
hmc
mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in
D. vulgaris
-mediated iron corrosion.
Tracing of Norovirus Outbreak Strains in Mussels Collected near Sewage Effluents
Authors: Nenonen Nancy P. , Hannoun Charles , Horal Peter , Hernroth Bodil , Bergström Tomas
Published: March 2008

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3′ (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.
Enterohemorrhagic
Escherichia coli
Exploits EspA Filaments for Attachment to Salad Leaves
Authors: Shaw Robert K. , Berger Cedric N. , Feys Bart , Knutton Stuart , Pallen Mark J. , Frankel Gad
Published: March 2008

Enterohemorrhagic
Escherichia coli
(EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.
Interactions between Food-Borne Pathogens and Protozoa Isolated from Lettuce and Spinach
Authors: Gourabathini Poornima , Brandl Maria T. , Redding Katherine S. , Gunderson John H. , Berk Sharon G.
Published: March 2008

The survival of
Salmonella enterica
was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of
Tetrahymena
. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with
S. enterica, Escherichia coli
O157:H7, and
Listeria monocytogenes. Glaucoma
sp.,
Colpoda steinii
, and
Acanthamoeba palestinensis
were cultured from store-bought spinach and lettuce and used in our study. A strain of
Tetrahymena pyriformis
previously isolated from spinach and a soil-borne
Tetrahymena
sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by
Glaucoma
with all of the bacterial strains, although
L. monocytogenes
resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of
Tetrahymena
on
E. coli
O157:H7 and
S. enterica
but not during grazing on
L. monocytogenes
, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast,
C. steinii
and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of
E. coli
O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.
Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting
Authors: Scheirlinck Ilse , Van der Meulen Roel , Van Schoor Ann , Vancanneyt Marc , De Vuyst Luc , Vandamme Peter , Huys Geert
Published: March 2008

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and
pheS
genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species
Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum
, and
Lactobacillus pontis
dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of
Acetobacter
sp. and a member of the
Erwinia
/
Enterobacter
/
Pantoea
group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.
Dramatic Activation of Antibiotic Production in
Streptomyces coelicolor
by Cumulative Drug Resistance Mutations
Authors: Wang Guojun , Hosaka Takeshi , Ochi Kozo
Published: March 2008

We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with
Streptomyces coelicolor
A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with
relA
disruption. This new approach, called “ribosome engineering,” requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.
Development of a Novel Screening Method for the Isolation of “
Cronobacter
” spp. (
Enterobacter sakazakii
)
Authors: Iversen Carol , Druggan Patrick , Schumacher Sandra , Lehner Angelika , Feer Claudia , Gschwend Karl , Joosten Han , Stephan Roger
Published: March 2008

A differential medium, “
Cronobacter
” screening broth, has been designed to complement agars based on hydrolysis of chromogenic α-glucopyranoside substrates. The broth was evaluated using 329
Enterobacteriaceae
strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples.
Indirect Transmission of
Escherichia coli
O157:H7 Occurs Readily among Swine but Not among Sheep
Authors: Cornick Nancy A. , VuKhac Hung
Published: March 2008

Transmission of
Escherichia coli
O157:H7 among reservoir animals is generally thought to occur either by direct contact between a naïve animal and an infected animal or by consumption of food or water containing the organism. Although ruminants are considered the major reservoir, there are two reports of human infections caused by
E. coli
O157:H7 linked to the consumption of pork products or to the contamination of fresh produce by swine manure. The objective of this study was to determine whether
E. coli
O157:H7 could be transmitted to naïve animals, both sheep and swine, that did not have any direct contact with an infected donor animal. We recovered
E. coli
O157:H7 from 10/10 pigs with nose-to-nose contact with the infected donor or animals adjacent to the donor and from 5/6 naïve pigs that were penned in the same room as the donor pig but 10 to 20 ft away. In contrast, when the experiment was repeated with sheep,
E. coli
O157:H7 was recovered from 4/6 animals that had nose-to-nose contact with the infected donor or adjacent animals and from 0/6 naïve animals penned 10 to 20 ft away from the donor. These results suggest that
E. coli
O157:H7 is readily transmitted among swine and that transmission can occur by the creation of contaminated aerosols.
Targeted Inactivation ofFrancisella tularensisGenes by Group II Introns
Authors: Rodriguez Stephen A. , Yu Jieh-Juen , Davis Greg , Arulanandam Bernard P. , Klose Karl E.
Published: March 2008

Studies of the molecular mechanisms of pathogenesis ofFrancisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption inF. tularensisutilizing mobile group II introns (targetrons) specifically optimized forF. tularensis. Utilizing a targetron targeted toblaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies:F. tularensissubsp.tularensis, F. tularensissubsp.holarctica, and “F. tularensissubsp.novicida.” A targetron was also utilized to inactivateF. tularensissubsp.holarctica iglC, a gene required for virulence. TheiglCgene is located within theFrancisellapathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, theiglCtargetron targeted both copies simultaneously, resulting in a strain mutated in bothiglCgenes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.
Identification of Hexose Transporter-Like Sensor
HXS1
and Functional Hexose Transporter
HXT1
in the Methylotrophic Yeast
Hansenula polymorpha
Authors: Stasyk Olena G. , Maidan Mykola M. , Stasyk Oleh V. , Van Dijck Patrick , Thevelein Johan M. , Sibirny Andriy A.
Published: March 2008

We identified in the methylotrophic yeast
Hansenula polymorpha
(syn.
Pichia angusta
) a novel hexose transporter homologue gene,
HXS1
(
h
e
x
ose
s
ensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene,
HXT1
(
h
e
x
ose
t
ransporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the
Saccharomyces cerevisiae
transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an
S. cerevisiae
hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host,
HXS1
is expressed at moderately low level and is required for glucose induction of the
H. polymorpha
functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However,
HXS1
deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the
hxs1
Δ deletion strain. Our combined results suggest that in the Crabtree-negative yeast
H. polymorpha
, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.
Unconventional Genomic Architecture in the Budding Yeast
Saccharomyces cerevisiae
Masks the Nested Antisense Gene
NAG1
Authors: Ma Jun , Dobry Craig J. , Krysan Damian J. , Kumar Anuj
Published: March 2008

The genomic architecture of the budding yeast
Saccharomyces cerevisiae
is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene,
NAG1
(for
n
ested
a
ntisense
g
ene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand.
NAG1
encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by β-galactosidase analysis of a
NAG1-lacZ
fusion.
NAG1
is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (
nag1
-
1
) disruptive for
NAG1
but silent with respect to YGR031W, defines a role for
NAG1
in yeast cell wall biogenesis; microarray profiling of
nag1
-
1
indicates decreased expression of genes contributing to cell wall organization, and the
nag1
-
1
mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus,
NAG1
is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.
Microneme Rhomboid Protease TgROM1 Is Required for Efficient Intracellular Growth of
Toxoplasma gondii
Authors: Brossier Fabien , Starnes G. Lucas , Beatty Wandy L. , Sibley L. David
Published: March 2008

Rhomboids are serine proteases that cleave their substrates within the transmembrane domain.
Toxoplasma gondii
contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments.
Toxoplasma
rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in
Plasmodium falciparum
processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of
T. gondii
. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of
TgROM1
using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type
T. gondii
. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by
T. gondii
.
Anastomosis Is Required for Virulence of the Fungal NecrotrophAlternaria brassicicola
Authors: Craven Kelly D. , Vélëz Heriberto , Cho Yangrae , Lawrence Christopher B. , Mitchell Thomas K.
Published: March 2008

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogenAlternaria brassicicolaand two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of theA. brassicicolaisolates tested, as well asA. mimiculaandA. japonica, were capable of self-fusion, with two isolates ofA. brassicicolabeing capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) inA. brassicicolaresulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenicA. brassicicolamutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity inA. brassicicola.
The Peptidoglycan-Associated Lipoprotein OprL Helps Protect a
Pseudomonas aeruginosa
Mutant Devoid of the Transactivator OxyR from Hydrogen Peroxide-Mediated Killing during Planktonic and Biofilm Culture
Authors: Panmanee Warunya , Gomez Francisco , Witte David , Pancholi Vijay , Britigan Bradley E. , Hassett Daniel J.
Published: March 2008

OxyR controls H
2
O
2
-dependent gene expression in
Pseudomonas aeruginosa
. Without OxyR, diluted (<10
7
/ml) organisms are easily killed by micromolar H
2
O
2
. The goal of this study was to define proteins that contribute to
oxyR
mutant survival in the presence of H
2
O
2
. We identified proteins in an
oxyR
mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double
oxyR oprL
mutant was constructed and was found to be more sensitive to H
2
O
2
than the
oxyR
mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H
2
O
2
. Given the sensitivity of
oxyR oprL
bacteria to planktonic H
2
O
2
, we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H
2
O
2
-mediated killing. Surprisingly, biofilm-grown
oxyR oprL
mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the
oxyR
mutant, were sensitive to killing by as little as 0.5 mM H
2
O
2
. Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of
oxyR
and
oxyR oprL
mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H
2
O
2
in the exquisitely sensitive
oxyR
mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.
aro
Mutations in
Salmonella enterica
Cause Defects in Cell Wall and Outer Membrane Integrity
Authors: Sebkova Alena , Karasova Daniela , Crhanova Magdalena , Budinska Eva , Rychlik Ivan
Published: March 2008

In this study we characterized
aro
mutants of
Salmonella enterica
serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the
aroA
,
aroD
, and
aroC
mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate
aro
gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the
aroD
mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of
murA
. To confirm this, we artificially overexpressed
murA
from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of
aro
mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.
Identification of AglE, a Second Glycosyltransferase Involved in N Glycosylation of the
Haloferax volcanii
S-Layer Glycoprotein
Authors: Abu-Qarn Mehtap , Giordano Assunta , Battaglia Francesca , Trauner Andrej , Hitchen Paul G. , Morris Howard R. , Dell Anne , Eichler Jerry
Published: March 2008

Archaea
, like
Eukarya
and
Bacteria
, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in
Archaea
. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the
Haloferax volcanii
S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in
Archaea
.
Identification of Novel Genes and Pathways Affecting
Salmonella
Type III Secretion System 1 Using a Contact-Dependent Hemolysis Assay
Authors: Field Terry R. , Layton Abigail N. , Bispham Jennie , Stevens Mark P. , Galyov Edouard E.
Published: March 2008

We screened 5,700
Salmonella enterica
serovar Typhimurium mutants for defects in type III secretion system 1 (T3SS-1)-mediated contact-dependent hemolysis to identify novel genes and pathways affecting the activity of T3SS-1. Our data suggest that previously unrecognized factors such as type I fimbriae may modulate the expression, activity, or deployment of this key virulence factor.
SOS Induction by Stabilized Topoisomerase IA Cleavage Complex Occurs via the RecBCD Pathway
Authors: Sutherland Jeanette H. , Cheng Bokun , Liu I-Fen , Tse-Dinh Yuk-Ching
Published: March 2008

Accumulation of mutant topoisomerase I cleavage complex can lead to SOS induction and cell death in
Escherichia coli
. The single-stranded break associated with mutant topoisomerase I cleavage complex is converted to double-stranded break, which then is processed by the RecBCD pathway, followed by association of RecA with the single-stranded DNA.