bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of window delay, delinearization, and frequency on tone-burst otoacoustic emission input/output measurements
Authors: Epstein Michael , Buus Søren , Florentine Mary
Published: August 2004

Tone-burst otoacoustic emissions (TBOAEs) are a potential tool for objectively examining cochlear activity in humans. However, their use requires knowledge of how the TBOAE input/output depends on measurement and analysis paradigms. The present experiment examined the effect of variations in response-window timing, response delinearization, and local changes in stimulus frequency on TBOAE input/output measurement. None of these experimental manipulations had a profound effect on TBOAE measurements as long as reasonable parameter choices were made. Nonetheless, judicial choice of the experimental parameters can optimize the assessment of BM I/O functions. It is concluded that the consistency of TBOAE I/O across the parameters tested makes it a viable tool to consider for examining human cochlear activity.
Mathematical model for collective bubble dynamics in strong ultrasound fields
Authors: Doinikov Alexander A.
Published: August 2004

Using the Lagrangian formalism, coupled equations are derived that describe radial and translational motions of an arbitrary bubble in a three-dimensional cluster subject to an ultrasound field. The obtained equations incorporate the nonlinear coupling of the volume and the translational modes of bubbles as well as interactions between all bubbles in the cluster up to terms of third order in the inverse separation distances. Solving these equations for each bubble in a multibubble structure, consisting of N bubbles with arbitrary spatial arrangement, the collective dynamics of this structure can be simulated. The potentialities of the proposed model in simulating the bubble dynamics in strong ultrasound fields are illustrated by numerical examples.
Sex differences in voice onset time: A developmental study of phonetic context effects in British English
Authors: Whiteside Sandra P. , Henry Luisa , Dobbin Rachel
Published: August 2004

Voice onset time (VOT) data for the plosives /p b t d k g/ in two vowel contexts (/i ɑ/) for 5 groups of 46 boys and girls aged 5; 8 (5 years, 8 months) to 13;2 years were investigated to examine patterns of sex differences. Results indicated that there was some evidence of females displaying longer VOT values than the males. In addition, these were found to be most marked for the data of the 13;2-year olds. Furthermore, the sex differences in the VOT values displayed phonetic context effects. For example, the greatest sex differences were observed for the voiceless plosives, and within the context of the vowel /i/.
Amplification and spectral shifts of vocalizations inside burrows of the frog Eupsophus calcaratus (Leptodactylidae)
Authors: Penna Mario
Published: August 2004

A variety of animals that communicate by sound emit signals from sites favoring their propagation, thereby increasing the range over which these sounds convey information. A different significance of calling sites has been reported for burrowing frogs Eupsophus emiliopugini from southern Chile: the cavities from which these frogs vocalize amplify conspecific vocalizations generated externally, thus providing a means to enhance the reception of neighbor’s vocalizations in chorusing aggregations. In the current study the amplification of vocalizations of a related species, E. calcaratus, is investigated, to explore the extent of sound enhancement reported previously. Advertisement calls broadcast through a loudspeaker placed in the vicinity of a burrow, monitored with small microphones, are amplified by up to 18 dB inside cavities relative to outside. The fundamental resonant frequency of burrows, measured with broadcast noise and pure tones, ranges from 842 to 1836 Hz and is significantly correlated with the burrow’s length. Burrows change the spectral envelope of incoming calls by increasing the amplitude of lower relative to higher harmonics. The call amplification effect inside burrows of E. calcaratus parallels the effect reported previously for E. emiliopugini, and indicates that the acoustic properties of calling sites may affect signal reception by burrowing animals.
Multimode shunt damping of piezoelectric smart panel for noise reduction
Authors: Kim Jaehwan , Kim Joon-Hyoung
Published: August 2004

Multimode shunt damping of piezoelectric smart panel is studied for noise reduction. Piezoelectric smart panel is a plate structure on which a piezoelectric patch is attached with an electrical shunt circuit. When an incidence sound is impinged on the panel structure, the structure vibrates and the attached piezoelectric patch produces an electrical energy, which can be effectively dissipated as heat via the electrical shunt circuit. Since the energy dissipation strongly depends on the vibration mode of the panel structure, many patches are required for multiple vibration modes. Instead of using multiple piezoelectric patches, a single piezoelectric patch is used in conjunction with a blocked shunt circuit for multimode shunt damping. Modeling, shunt parameter tuning, and implementation of the blocked shunt circuit along with an acoustic test of the panel are explained. A remarkable reduction of the transmitted noise was achieved for multiple modes of the panel. Since this technology has many merits in terms of compactness, low cost, robustness, and ease of installation, practical applications in many noise problems can be anticipated.
Iterative algorithms for computing the shape of a hard scattering object: Computing the shape derivative
Authors: Norton Stephen J.
Published: August 2004

The problem of determining the shape of an acoustically hard scattering object from remote scattering measurements is considered. An iterative approach is used to find the object shape that minimizes the mean-squared difference between a set of actual and predicted scattering observations. A crucial task in this minimization is the computation of the “shape derivative,” or functional gradient, of the mean-square error with respect to the object’s shape or boundary. The shape derivative tells us how to update the object’s shape to reduce the mean-square error at each iteration. If, for example, the object’s boundary is parameterized with N variables, a brute-force approach to computing the shape derivative using finite-differences would require a minimum of N+1 forward solutions per iteration. We show how the shape derivative can be computed with just two forward solutions: one ordinary forward solution and a suitably constructed adjoint solution. This approach is independent of N and is not only far more efficient, but numerically less error prone, than finite-difference schemes for computing derivatives.
Relationship of neural and otoacoustic emission thresholds during endocochlear potential development in the gerbil
Authors: Mills David M.
Published: August 2004

Distortion product otoacoustic emissions and auditory brainstem responses (ABRs) were measured in neonatal gerbils at three ages: at 15–16 days after birth (dab), near the onset of hearing when the endocochlear potential (EP) is known to be still immature; at 22 dab, when the EP first reaches mature levels; and at 30 dab. Comparing individual 15–16 dab animals to the 22 dab group, ABR threshold changes were typically larger than those for cubic distortion tone (CDT, 2f1−f2) emission thresholds which were, in turn, larger than those for the simple difference tone (DT, f2−f1). In contrast, from 22 to 30 dab there were no important changes in CDT or DT emission thresholds. Observed threshold-change relationships were very similar to those found in differential diagnosis investigations, where the EP was experimentally decreased using a chronic furosemide application. Therefore, most of the change in cochlear function over the two week period studied could be attributed to the maturation of EP during the first week. Model calculations further show that relative changes in CDT and DT emission thresholds are compatible with a movement of the operating point of the cochlear amplifier toward its symmetrical “central” point as the EP reaches mature levels.
Acoustic thermometry of the western Mediterranean basin
Authors: Skarsoulis E. K. , Send U. , Piperakis G. , Testor P.
Published: August 2004

Ocean acoustic tomography is used to obtain heat-content estimates for the western Mediterranean basin. Travel-time data from 13 tomography sections of the Thetis-2 experiment (January–October 1994) are analyzed with a matched-peak inversion approach. The underlying analysis involves the use of peak arrivals and nonlinear model relations between travel-time and sound-speed variations. Slice inversion results are combined with temperature covariance functions for the western Mediterranean to obtain heat-content estimates for the basin. These estimates compare favorably with ECMWF data over the nine-month period of the Thetis-2 experiment. Furthermore, estimates for the basin-average temperature of the western Mediterranean deep water are obtained.
Acoustic waves generated by a laser point source in an isotropic cylinder
Authors: Pan Yongdong , Rossignol Clément , Audoin Bertrand
Published: August 2004

The acoustic field of a homogeneous and isotropic cylinder generated by a laser point source in either ablation or thermoelastic regime is obtained theoretically. A three-dimensional Fourier transform is used to calculate the acoustic displacement at the cylinder surface. Experimental waveforms were measured and analyzed for both regimes. Theoretical normal displacements under either regime are calculated and compared to the experimental signals for aluminum cylinders. Very good agreements are observed in the arrival time, shape, and relative amplitude (i) of the cylindrical Rayleigh waves with different round trips, and (ii) of the various longitudinal and transverse bulk waves propagating through the cylinder or reflected at the free circular surface.
Chronic excitotoxicity in the guinea pig cochlea induces temporary functional deficits without disrupting otoacoustic emissions
Authors: Le Prell Colleen G. , Yagi Masao , Kawamoto Kohei , Beyer Lisa A. , Atkin Graham , Raphael Yehoash , Dolan David F. , Bledsoe Sanford C. , Moody David B.
Published: August 2004

Brief cochlear excitotoxicity produces temporary neural swelling and transient deficits in auditory sensitivity; however, the consequences of long-lasting excitotoxic insult have not been tested. Chronic intra-cochlear infusion of the glutamate agonist AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) resulted in functional deficits in the sound-evoked auditory brainstem response, as well as in behavioral measures of hearing. The electrophysiological deficits were similar to those observed following acute infusion of AMPA into the cochlea; however, the concentration-response curve was significantly shifted as a consequence of the slower infusion rate used with chronic cochlear administration. As observed following acute excitotoxic insult, complete functional recovery was evident within 7 days of discontinuing the AMPA infusion. Distortion product otoacoustic emissions were not affected by chronic AMPA infusion, suggesting that trauma to outer hair cells did not contribute to AMPA-induced deficits in acoustic sensitivity. Results from the current experiment address the permanence of deficits induced by chronic (14 day) excitotoxic insult as well as deficits in psychophysical detection of longer duration acoustic signals.
Improved algorithms and methods for room sound-field prediction by acoustical radiosity in arbitrary polyhedral rooms
Authors: Nosal Eva-Marie , Hodgson Murray , Ashdown Ian
Published: August 2004

This paper explores acoustical (or time-dependent) radiosity—a geometrical-acoustics sound-field prediction method that assumes diffuse surface reflection. The literature of acoustical radiosity is briefly reviewed and the advantages and disadvantages of the method are discussed. A discrete form of the integral equation that results from meshing the enclosure boundaries into patches is presented and used in a discrete-time algorithm. Furthermore, an averaging technique is used to reduce computational requirements. To generalize to nonrectangular rooms, a spherical-triangle method is proposed as a means of evaluating the integrals over solid angles that appear in the discrete form of the integral equation. The evaluation of form factors, which also appear in the numerical solution, is discussed for rectangular and nonrectangular rooms. This algorithm and associated methods are validated by comparison of the steady-state predictions for a spherical enclosure to analytical solutions.
An Active Type IV Secretion System Encoded by the F Plasmid SensitizesEscherichia colito Bile Salts
Authors: Bidlack James E. , Silverman Philip M.
Published: August 2004

F+strains ofEscherichia coliinfected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F+strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F−strain or as a reduced plating efficiency of the F+strain on solid media; at 2% bile salts, plating efficiency was reduced 104-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F+cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F−cells, suggesting a selectivity for amphipathic anions. A mutation in any but one Ftragene required for the assembly of F pili, including thetraAgene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception wastraW. AtraWmutant was 100-fold more sensitive to cholate than thetra+strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F+cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic β-lactamase into the medium.
Genetic Manipulation of Carotenoid Biosynthesis in the Green Sulfur Bacterium
Chlorobium tepidum
Authors: Frigaard Niels-Ulrik , Maresca Julia A. , Yunker Colleen E. , Jones A. Daniel , Bryant Donald A.
Published: August 2004

The green sulfur bacterium
Chlorobium tepidum
is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of
C. tepidum
, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (
crtB
/
CT1386
), phytoene desaturase (
crtP
/
CT0807
), ζ-carotene desaturase (
crtQ
/
CT1414
), γ-carotene desaturase (
crtU
/
CT0323
), carotenoid 1′,2′-hydratase (
crtC
/
CT0301
), and carotenoid
cis
-
trans
isomerase (
crtH
/
CT0649
). Three mutants (
CT0180
,
CT1357
, and
CT1416
mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in
C. tepidum
is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like
cis
-
trans
isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a
crtB
mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll
a
contents of mutants lacking colored carotenoids (
crtB
,
crtP
, and
crtQ
mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in
C. tepidum
offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.
The CcpA Protein Is Necessary for Efficient Sporulation and Enterotoxin Gene (cpe) Regulation inClostridium perfringens
Authors: Varga John , Stirewalt Veronica L. , Melville Stephen B.
Published: August 2004

Clostridium perfringensis the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production ofC. perfringensenterotoxin encoded by thecpegene. Glucose is a catabolite repressor of sporulation byC. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into theccpAgene ofC. perfringensby conjugational transfer of a nonreplicating plasmid intoC. perfringens, which led to inactivation of theccpAgene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of whichC. perfringensis a member. TheccpAmutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in theccpAmutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation inC. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of thecpegene was measured in the wild-type andccpAmutant strains grown in sporulation medium by using acpe-gusAfusion (gusAis anEscherichia coligene encoding the enzyme β-glucuronidase). In the exponential growth phase,cpetranscription was two times higher in theccpAmutant strain than in the wild-type strain. Transcription ofcpewas highly induced during the entry into stationary phase in wild-type cells but was not induced in theccpAmutant strain. Glucose repressedcpetranscription in both the wild-type andccpAmutant strain. Therefore, CcpA appears to act as a repressor ofcpetranscription in exponential growth but is required for efficient sporulation andcpetranscription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).
Differences in Enzymatic Properties Allow SodCI but Not SodCII To Contribute to Virulence in
Salmonella enterica
Serovar Typhimurium Strain 14028
Authors: Krishnakumar Radha , Craig Maureen , Imlay James A. , Slauch James M.
Published: August 2004

Salmonella enterica
serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in
sodCI
attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a
sodCI
background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of
sodCI
and
sodCII
, we found that SodCI contributes to virulence when placed under the control of the
sodCII
promoter. In contrast, SodCII does not contribute to virulence even when expressed from the
sodCI
promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is “tethered” within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.
Comparative Genetic Diversity of
Pseudomonas stutzeri
Genomovars, Clonal Structure, and Phylogeny of the Species
Authors: Cladera Aina Maria , Bennasar Antonio , Barceló Maria , Lalucat Jorge , García-Valdés Elena
Published: August 2004

A combined phylogenetic and multilocus DNA sequence analysis of 26
Pseudomonas stutzeri
strains distributed within the 9 genomovars of the species has been performed. Type strains of the two most closely related species (
P. balearica
, former genomovar 6, and
P. mendocina
), together with
P. aeruginosa
, as the type species of the genus, have been included in the study. The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis. Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1,
gyrB
,
rpoD
,
nosZ
,
catA
, and
nahH
) confirmed the monophyletic origin of the genomovars within the
Pseudomonas
branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species.
TheEscherichia coliGTPase CgtAECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase
Authors: Wout P. , Pu K. , Sullivan S. M. , Reese V. , Zhou S. , Lin B. , Maddock J. R.
Published: August 2004

CgtAE/ObgE/YhbZ is anEscherichia coliguanine nucleotide binding protein of the Obg/GTP1 subfamily whose members have been implicated in a number of cellular functions including GTP-GDP sensing, sporulation initiation, and translation. Here we describe a kinetic analysis of CgtAEwith guanine nucleotides and show that its properties are similar to those of theCaulobacter crescentushomolog CgtAC. CgtAEbinds both GTP and GDP with moderate affinity, shows high guanine nucleotide exchange rate constants for both nucleotides, and has a relatively low GTP hydrolysis rate. We show that CgtAEis associated predominantly with the 50S ribosomal subunit. Interestingly, CgtAEcopurifies with SpoT, a ribosome-associated ppGpp hydrolase/synthetase involved in the stress response. The interaction between CgtAEand SpoT was confirmed by reciprocal coprecipitation experiments and by two-hybrid assays. These studies raise the possibility that the ribosome-associated CgtAEis involved in the SpoT-mediated stress response.
Control of Virulence by the Two-Component System CiaR/H Is Mediated via HtrA, a Major Virulence Factor ofStreptococcus pneumoniae
Authors: Ibrahim Yasser Musa , Kerr Alison R. , McCluskey Jackie , Mitchell Tim J.
Published: August 2004

The CiaR/H two-component system is involved in regulating virulence and competence inStreptococcus pneumoniae. The system is known to regulate many genes, including that for high-temperature requirement A (HtrA). This gene has been implicated in the ability of the pneumococcus to colonize the nasopharynx of infant rats. We reported previously that deletion of the gene for HtrA made the pneumococcal strains much less virulent in mouse models, less able to grow at higher temperatures, and more sensitive to oxidative stress. In this report, we show that the growth phenotype as well as sensitivity to oxidative stress of ΔciaRmutant was very similar to that of a ΔhtrAmutant and that the expression of the HtrA protein was reduced in aciaR-null mutant. Both the in vitro phenotype and the reduced virulence of ΔciaRmutant could be restored by increasing the expression of HtrA.
Identification ofsarV(SA2062), a New Transcriptional Regulator, Is Repressed by SarA and MgrA (SA0641) and Involved in the Regulation of Autolysis inStaphylococcus aureus
Authors: Manna Adhar C. , Ingavale Susham S. , Maloney MaryBeth , van Wamel Willem , Cheung Ambrose L.
Published: August 2004

The expression of genes involved in the pathogenesis ofStaphylococcus aureusis known to be controlled by global regulatory loci, includingagr,sarA,sae,arlRS,lytSR, andsarA-like genes. Here we described a novel transcriptional regulator calledsarVof the SarA protein family. The transcription ofsarVis low or undetectable under in vitro conditions but is significantly augmented insarAandmgrA(norRorrat) (SA0641) mutants. ThesarAandmgrAgenes act as repressors ofsarVexpression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of thesarVpromoter as determined by gel shift and DNase I footprinting assays. The expression of 19 potential target genes involved in autolysis and virulence, phenotypes affected bysarAandmgrA, was evaluated in an isogenicsarVmutant pair. Our data indicated that thesarVgene product played a role regulating some virulence genes and more genes involved in autolysis. ThesarVmutant was more resistant to Triton X-100 and penicillin-induced lysis compared to the wild type and thesarAmutant, whereas hyperexpression ofsarVin the parental strain or thesarVmutant rendered the resultant strain highly susceptible to lysis. Zymographic analysis of murein hydrolase activity revealed that inactivation of thesarVgene results in decreased extracellular murein hydrolase activity compared to that of wild-typeS. aureus. We propose thatsarVmay be part of the common pathway by whichmgrAandsarAgene products control autolysis inS. aureus.
Interaction of PomB with the Third Transmembrane Segment of PomA in the Na
+
-Driven Polar Flagellum of
Vibrio alginolyticus
Authors: Yakushi Toshiharu , Maki Shingo , Homma Michio
Published: August 2004

The marine bacterium
Vibrio alginolyticus
has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na
+
-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na
+
than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na
+
conduction to flagellar rotation.
Biochemical Characterization of StyAB from
Pseudomonas
sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase
Authors: Otto Katja , Hofstetter Karin , Röthlisberger Martina , Witholt Bernard , Schmid Andreas
Published: August 2004

Pseudomonas
sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type
Pseudomonas
strain VLB120 as well as from recombinant
Escherichia coli
. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C═C double bonds. Styrene is exclusively converted to
S
-styrene oxide with a specific activity of 2.1 U mg
−1
(
k
cat
= 1.6 s
−1
) and

K
m

values for styrene of 0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg
−1
(
k
cat
[NADH] = 60 s
−1
). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.
Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of
Escherichia coli
K-12
Authors: Sauter Annette , Braun Volkmar
Published: August 2004

The FecA outer membrane protein of
Escherichia coli
functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the
fec
transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the β-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the β-barrel and to contribute to the fixation of the globular domain inside the β-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.
Molecular Analysis of Cytolysin A (ClyA) in Pathogenic
Escherichia coli
Strains
Authors: Ludwig Albrecht , von Rhein Christine , Bauer Susanne , Hüttinger Christian , Goebel Werner
Published: August 2004

Cytolysin A (ClyA) of
Escherichia coli
is a pore-forming hemolytic protein encoded by the
clyA
(
hlyE
,
sheA
) gene that was first identified in
E. coli
K-12. In this study we examined various clinical
E. coli
isolates with regard to the presence and integrity of
clyA
. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing
E. coli
(STEC) strains, all 7 tested enteroinvasive
E. coli
(EIEC) strains, 6 of 8 enteroaggregative
E. coli
(EAEC) strains, and 4 of 7 tested enterotoxigenic
E. coli
(ETEC) strains possess a complete
clyA
gene. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic
E. coli
(EPEC) strains were shown to harbor mutant
clyA
derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence. The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated
E. coli
strains (
n
= 14 and 3, respectively) carried only nonfunctional
clyA
fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the
clyA
locus. Expression of
clyA
from clinical
E. coli
isolates proved to be positively controlled by the transcriptional regulator SlyA. Several tested
E. coli
strains harboring a functional
clyA
gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a
clyA
-dependent hemolytic phenotype only when SlyA was overexpressed. The presented data indicate that cytolysin A can play a role only for some of the pathogenic
E. coli
strains.
The Usher N Terminus Is the Initial Targeting Site for Chaperone-Subunit Complexes and Participates in Subsequent Pilus Biogenesis Events
Authors: Ng Tony W. , Akman Leyla , Osisami Mary , Thanassi David G.
Published: August 2004

Pilus biogenesis on the surface of uropathogenic
Escherichia coli
requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.
Role of an Inducible Single-Domain Hemoglobin in Mediating Resistance to Nitric Oxide and Nitrosative Stress in
Campylobacter jejuni
and
Campylobacter coli
Authors: Elvers Karen T. , Wu Guanghui , Gilberthorpe Nicola J. , Poole Robert K. , Park Simon F.
Published: August 2004

Campylobacter jejuni
expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from
Vitreoscilla stercoraria
and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of
C. jejuni
was hypersensitive to nitrosating agents (
S
-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress,
cgb
expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the
cgb
mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the
cgb
mutant. Based on similar studies in
Campylobacter coli
, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.
Penetration of Membrane-Containing Double-Stranded-DNA Bacteriophage PM2 intoPseudoalteromonasHosts
Authors: Kivelä Hanna M. , Daugelavičius Rimantas , Hankkio Riina H. , Bamford Jaana K. H. , Bamford Dennis H.
Published: August 2004

The icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of theCorticoviridaefamily. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marinePseudoalteromonashost has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.
Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors inStreptomyces ambofaciens
Authors: Roth Virginie , Aigle Bertrand , Bunet Robert , Wenner Thomas , Fourrier Céline , Decaris Bernard , Leblond Pierre
Published: August 2004

The duplicatedhasRandhasLgenes ofStreptomyces ambofaciensencode alternative sigma factors (named σBRand σBL) belonging to the σBgeneral stress response family inBacillus subtilis. The duplication appears to be the result of a recent event that occurred specifically inS. ambofaciens. The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level. In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent. WhilehasLis monocistronic,hasRis arranged in a polycistronic unit with two upstream open reading frames,arsRandprsR, that encode putative anti-anti-σ and anti-σ factors, respectively. Transcription of eachhasgene is initiated from two promoters. In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by theB. subtilisgeneral stress response sigma factor σB. Expression from this type of promoter for each of thehasgenes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses. Reverse transcription-PCR measurements indicate thathasRis 100 times more strongly expressed thanhasLfrom the σB-like promoter. Transcription from the second promoter of each gene (located upstream ofarsRin the case of thehasRlocus) appears to be constitutive and weak. Quantitative transcriptional analysis in single and doublehasmutant strains revealed that σBRand σBLdirect their own transcription as well as that of their duplicates. Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the σ factors implied in stress response inStreptomyces.
Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in
Yersinia enterocolitica
Authors: Burghout Peter , Beckers Frank , de Wit Emmie , van Boxtel Ria , Cornelis Guy R. , Tommassen Jan , Koster Margot
Published: August 2004

The YscC secretin is a major component of the type III protein secretion system of
Yersinia enterocolitica
and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in
trans
in a
Y. enterocolitica
strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ∼30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.
Bacillus subtilis
YhcR, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure
Authors: Oussenko Irina A. , Sanchez Roberto , Bechhofer David H.
Published: August 2004

In a continuing effort to identify ribonucleases that may be involved in mRNA decay in
Bacillus subtilis
, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3′-to-5′ exoribonucleases (polynucleotide phosphorylase [PNPase], RNase R, and YhaM) was undertaken. These experiments revealed the presence of a high-molecular-weight nuclease encoded by the
yhcR
gene that was active in the presence of Ca
2+
and Mn
2+
. YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3′-monophosphate products, similar to the well-characterized micrococcal nuclease of
Staphylococcus aureus
. YhcR appears to be located principally in the cell wall and is likely to be a substrate for a
B. subtilis
sortase. Zymogram analysis suggests that YhcR is the major Ca
2+
-activated nuclease of
B. subtilis
. In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named “NYD,” for “new YhcR domain.”
Indole-3-Acetic Acid Biosynthesis Is Deficient in
Gluconacetobacter diazotrophicus
Strains with Mutations in Cytochrome
c
Biogenesis Genes
Authors: Lee Sunhee , Flores-Encarnación M. , Contreras-Zentella M. , Garcia-Flores L. , Escamilla J. E. , Kennedy Christina
Published: August 2004

Gluconacetobacter diazotrophicus
is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of
G. diazotrophicus
unable to fix nitrogen, suggested that there are two beneficial effects of
G. diazotrophicus
on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by
G. diazotrophicus
, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn
5
mutants, released only ∼6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA
−
phenotype was not linked to Tn
5
. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome
c
biogenesis (
ccm
, for cytochrome
c
maturation). The
G. diazotrophicus ccm
operon was sequenced; the individual
ccm
gene products were 37 to 52% identical to
ccm
gene products of
Escherichia coli
and equivalent
cyc
genes of
Bradyrhizobium japonicum
. Although several
ccm
mutant phenotypes have been described in the literature, there are no reports of
ccm
gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the
ccm
genes of
G. diazotrophicus
are involved in cytochrome
c
biogenesis.
Kinetic Analysis of tRNA-Directed Transcription Antitermination of the
Bacillus subtilis glyQS
Gene In Vitro
Authors: Grundy Frank J. , Henkin Tina M.
Published: August 2004

Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNA
Gly
-dependent antitermination of the
Bacillus subtilis glyQS
leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the
glyQS
leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNA
Gly
can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.