bluuebunny/crossref_metadata_2025 huggingface dataset:
A modeling investigation of articulatory variability and acoustic stability during American English /r/ production
Authors: Nieto-Castanon Alfonso , Guenther Frank H. , Perkell Joseph S. , Curtin Hugh D.
Published: May 2005

This paper investigates the functional relationship between articulatory variability and stability of acoustic cues during American English /r/ production. The analysis of articulatory movement data on seven subjects shows that the extent of intrasubject articulatory variability along any given articulatory direction is strongly and inversely related to a measure of acoustic stability (the extent of acoustic variation that displacing the articulators in this direction would produce). The presence and direction of this relationship is consistent with a speech motor control mechanism that uses a third formant frequency (F3) target; i.e., the final articulatory variability is lower for those articulatory directions most relevant to determining the F3 value. In contrast, no consistent relationship across speakers and phonetic contexts was found between hypothesized vocal-tract target variables and articulatory variability. Furthermore, simulations of two speakers’ productions using the DIVA model of speech production, in conjunction with a novel speaker-specific vocal-tract model derived from magnetic resonance imaging data, mimic the observed range of articulatory gestures for each subject, while exhibiting the same articulatory/acoustic relations as those observed experimentally. Overall these results provide evidence for a common control scheme that utilizes an acoustic, rather than articulatory, target specification for American English /r/.
Simulations of optoacoustic wave propagation in light-absorbing media using a finite-difference time-domain method
Authors: Huang Deng-Huei , Liao Chao-Kang , Wei Chen-Wei , Li Pai-Chi
Published: May 2005

Optoacoustic (OA) imaging is an emerging technology that combines the high optical contrast of tissues with the high spatial resolution of ultrasound. Taking full advantage of OA imaging requires a better understanding of OA wave propagation in light-absorbing media. Current simulation methods are mainly based on simplified conditions such as thermal confinement, negligible viscosity, and homogeneous acoustic properties throughout the image object. In this study a new numerical approach is proposed based on a finite-difference time-domain (FDTD) method to solve the general OA equations, comprising the continuity, Navier-Stokes, and heat-conduction equations. The FDTD code was validated using a benchmark problem that has an approximate analytical solution. OA experiments were also conducted and data were in good agreement with those predicted by the FDTD method. Characteristics of simulated OA waveforms and OA images were discussed. The simulator was also employed to study wavefront distortion in OA breast imaging.
Off-axis sonar beam pattern of free-ranging finless porpoises measured by a stereo pulse event data logger
Authors: Akamatsu Tomonari , Wang Ding , Wang Kexiong
Published: May 2005

The off-axis sonar beam patterns of eight free-ranging finless porpoises were measured using attached data logger systems. The transmitted sound pressure level at each beam angle was calculated from the animal’s body angle, the water surface echo level, and the swimming depth. The beam pattern of the off-axis signals between 45° and 115° (where 0° corresponds to the on-axis direction) was nearly constant. The sound pressure level of the off-axis signals reached 162 dB re 1 μPa peak-to-peak. The surface echo level received at the animal was over 140 dB, much higher than the auditory threshold level of small odontocetes. Finless porpoises are estimated to be able to receive the surface echoes of off-axis signals even at 50-m depth. Shallow water systems (less than 50-m depth) are the dominant habitat of both oceanic and freshwater populations of this species. Surface echoes may provide porpoises not only with diving depth information but also with information about surface direction and location of obstacles (including prey items) outside the on-axis sector of the sonar beam.
Exploring interaction effects in two-component gas mixtures using orthogonal signal correction of ultrasound pulses
Authors: Carlson Johan E. , Martinsson Pär-Erik
Published: May 2005

Within Sweden and the EU, an increased use of biogas gas and natural gas is encouraged to decrease emission of carbon dioxide. To support more effective manufacturing, distribution, and consumption of energy gases, new methods for the measurement of the calorimetric value or the gas composition are needed. This paper presents a method to extract and visualize variations in ultrasound pulse shape, caused by interaction effects between the constituents of a two-component gas mixture. The method is based on a combination of principal component analysis and orthogonal signal correction. Pulse-echo ultrasound experiments on mixtures of oxygen and ethane in the concentration range from 20% to 80% ethane show that the extracted information could be correlated with the molar fraction of ethane in the mixture.
Acoustic analysis in Mudejar-Gothic churches: Experimental results
Authors: Galindo Miguel , Zamarreño Teófilo , Girón Sara
Published: May 2005

This paper describes the preliminary results of research work in acoustics, conducted in a set of 12 Mudejar-Gothic churches in the city of Seville in the south of Spain. Despite common architectural style, the churches feature individual characteristics and have volumes ranging from 3947 to 10 708 m3. Acoustic parameters were measured in unoccupied churches according to the ISO-3382 standard. An extensive experimental study was carried out using impulse response analysis through a maximum length sequence measurement system in each church. It covered aspects such as reverberation (reverberation times, early decay times), distribution of sound levels (sound strength); early to late sound energy parameters derived from the impulse responses (center time, clarity for speech, clarity, definition, lateral energy fraction), and speech intelligibility (rapid speech transmission index), which all take both spectral and spatial distribution into account. Background noise was also measured to obtain the NR indices. The study describes the acoustic field inside each temple and establishes a discussion for each one of the acoustic descriptors mentioned by using the theoretical models available and the principles of architectural acoustics. Analysis of the quality of the spaces for music and speech is carried out according to the most widespread criteria for auditoria.
Normal modes of a single mandolin with different bracing patterns installed
Authors: Cohen David J. , Rossing Thomas D.
Published: May 2005

The vibrational modes and sound spectra of a single archtop mandolin constructed with a removable back plate were studied with different bracing patterns installed. The bracing patterns were (i) symmetric and (ii) asymmetric parallel ‘‘tone bars,’’ (iii) X-bracing, and (iv) an asymmetric pattern previously used and studied [D. Cohen and T. D. Rossing, Can. Aeronautics Space, J. 4(2), 48–54 (2000), D. Cohen and T. D. Rossing, Acoust. Sci. Tech. 24, 1–6 (2003)]. The results have been compared with those obtained previously for vintage mandolins [D. Cohen and T. D. Rossing, ‘‘Normal modes of different types of pre-1929 mandolins,’’ presented at the 147th meeting of the Acoustical Society of America, New York, NY (2004)] and for mandolins constructed more recently. As was the case for the different types of vintage mandolins, changing the bracing pattern has little effect on the shapes of the normal modes, but does affect the frequencies of the normal modes. The X- or crossed bracing pattern imparts more cross-grain stiffness than the other bracing patterns, with the result that modes involving cross-grain bending occur at higher frequencies than is the case for the other bracing patterns.
Progress towards an electro-acoustic resonance technique for determining quantitative material and geometrical properties in high contrast multi-layer elastic structures.
Authors: Fisher Karl
Published: May 2005

High contrast multilayered elastic structures continue to be problematic for ultrasonic inspection. Large acoustic material impedance mismatches, refraction, reverberation, multiple echoes, and high elastic attenuations are just some of the issues one is faced with standard high frequency (1–20 MHz) pulse echo detection and imaging methods. In this presentation, we will present progress towards developing a low frequency resonance technique that operates in the 20 to 70 kHz regime. The technique is based on a direct correlation between the electrical impedance of a standard electro-acoustic transducer and the mechanical loading it experiences when placed in contact with a layered elastic structure. Preliminary experimental and theoretical results are in good agreement.
Relationship of
DFG16
to the Rim101p pH Response Pathway in
Saccharomyces cerevisiae
and
Candida albicans
Authors: Barwell Karen J. , Boysen Jacob H. , Xu Wenjie , Mitchell Aaron P.
Published: May 2005

Many fungal pH responses depend upon conserved Rim101p/PacC transcription factors, which are activated by C-terminal proteolytic processing. The means by which environmental pH is sensed by this pathway are not known. Here, we report a screen of the
Saccharomyces cerevisiae
viable deletion mutant library that has yielded a new gene required for processed Rim101p accumulation,
DFG16.
An
S. cerevisiae dfg16
Δ mutant expresses Rim101p-repressed genes at elevated levels. In addition,
Candida albicans dfg16
Δ/
dfg16Δ
mutants are defective in alkaline pH-induced filamentation, and their defect is suppressed by expression of truncated Rim101-405p. Thus, Dfg16p is a functionally conserved Rim101p pathway member. Many proteins required for processed Rim101p accumulation are members of the ESCRT complex, which functions in the formation of multivesicular bodies (MVBs). Staining with the dye FM4-64 indicates that the
S. cerevisiae dfg16
Δ mutant does not have an MVB defect. We find that two transcripts,
PRY1
and
ASN1
, respond to mutations that affect both the Rim101p and MVB pathways but not to mutations that affect only one pathway. The
S. cerevisiae dfg16
Δ mutation does not affect
PRY1
and
ASN1
expression, thus confirming that Dfg16p function is restricted to the Rim101p pathway. Dfg16p is homologous to
Aspergillus nidulans
PalH, a component of the well-characterized PacC processing pathway. We verify that the previously recognized PalH homolog, Rim21p, also functions in the
S. cerevisiae
Rim101p pathway. Dfg16p is predicted to have seven membrane-spanning segments and a long hydrophilic C-terminal region, as expected if Dfg16p were a G-protein-coupled receptor.
Molecular and Functional Analyses of
poi-2
, a Novel Gene Highly Expressed in Sexual and Perithecial Tissues of
Neurospora crassa
Authors: Kim Hyojeong , Nelson Mary Anne
Published: May 2005

The
poi-2
gene is highly and specifically expressed in starved and sexual tissues of the filamentous fungus
Neurospora crassa
. It encodes a 27-kDa protein, as shown by in vitro transcription and translation. The POI2 protein contains a hydrophobic signal sequence at the amino terminus followed by novel 16 tandem repeats of 13 to 14 amino acid residues; all repeats are separated by Kex2 processing sites. Repeat-induced point mutation (RIP)-mediated gene disruption was used to generate
poi-2
mutants, and the mutated sequences showed either one of two distinct patterns: typical RIPs (GC-to-AT transitions) or insertion-deletion (indel) mutations. Although the
poi-2
strains contained numerous mutations, all retained intact open reading frames (ORFs) of various lengths. They showed greatly reduced vegetative growth and protoperithecial formation and low viability of their sexual progeny. All
poi-2
mutants had similar defects in male fertility and the mating response, but the nature of female fertility defects varied and corresponded to the length of the residual
poi-2
ORF. Mutants with ORFs of approximately normal length occasionally completed sexual development and produced viable ascospores, while a mutant with a severely truncated ORF was female sterile due to its inability to form protoperithecia. Thus,
poi-2
is essential for differentiation of female reproductive structures and perithecial development as well as for normal vegetative growth. The POI2 protein is involved in the mating response, probably as a component in the pathway rather than as a pheromone.
Protein Engineering of the Archetypal Nitroarene Dioxygenase of
Ralstonia
sp. Strain U2 for Activity on Aminonitrotoluenes and Dinitrotoluenes through Alpha-Subunit Residues Leucine 225, Phenylalanine 350, and Glycine 407
Authors: Keenan Brendan G. , Leungsakul Thammajun , Smets Barth F. , Mori Masa-aki , Henderson David E. , Wood Thomas K.
Published: May 2005

Naphthalene dioxygenase (NDO) from
Ralstonia
sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the α-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO
nagAcAd
also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.
Mosaic Prophages with Horizontally Acquired Genes Account for the Emergence and Diversification of the Globally Disseminated M1T1 Clone of
Streptococcus pyogenes
Authors: Aziz Ramy K. , Edwards Robert A. , Taylor William W. , Low Donald E. , McGeer Allison , Kotb Malak
Published: May 2005

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.
Persistence ofStreptococcus pyogenesin Stationary-Phase Cultures
Authors: Wood Daniel N. , Chaussee Michelle A. , Chaussee Michael S. , Buttaro Bettina A.
Published: May 2005

In addition to causing fulminant disease,Streptococcus pyogenesmay be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) ofS. pyogenes. When grown in sugar-limited Todd-Hewitt broth,S. pyogenescells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 103CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions,S. pyogenescells can persist for greater than 1 year as a dynamic population.
Transcription Activation In Vitro by the
Bradyrhizobium japonicum
Regulatory Protein FixK
2
Authors: Mesa Socorro , Ucurum Zöhre , Hennecke Hauke , Fischer Hans-Martin
Published: May 2005

In
Bradyrhizobium japonicum
, the N
2
-fixing root nodule endosymbiont of soybean, a group of genes required for microaerobic, anaerobic, or symbiotic growth is controlled by FixK
2
, a key regulator that is part of the FixLJ-FixK
2
cascade. FixK
2
belongs to the family of cyclic AMP receptor protein/fumarate and nitrate reductase (CRP/FNR) transcription factors that recognize a palindromic DNA motif (CRP/FNR box) associated with the regulated promoters. Here, we report on a biochemical analysis of FixK
2
and its transcription activation activity in vitro. FixK
2
was expressed in
Escherichia coli
and purified as a soluble N-terminally histidine-tagged protein. Gel filtration experiments revealed that increasing the protein concentration shifts the monomer-dimer equilibrium toward the dimer. Purified FixK
2
productively interacted with the
B. japonicum
σ
80
-RNA polymerase holoenzyme, but not with
E. coli
σ
70
-RNA polymerase holoenzyme, to activate transcription from the
B. japonicum fixNOQP
,
fixGHIS
, and
hemN
2
promoters in vitro. Furthermore, FixK
2
activated transcription from the
E. coli
FF(−41.5) model promoter, again only in concert with
B. japonicum
RNA polymerase. All of these promoters are so-called class II CRP/FNR-type promoters. We showed by specific mutagenesis that the FixK
2
box at nucleotide position −40.5 in the
hemN
2
promoter, but not that at −78.5, is crucial for activation both in vivo and in vitro, which argues against recognition of a potential class III promoter. Given the lack of any evidence for the presence of a cofactor in purified FixK
2
, we surmise that FixK
2
alone is sufficient to activate in vitro transcription to at least a basal level. This contrasts with all well-studied CRP/FNR-type proteins, which do require coregulators.
Plasmid Partition System of the P1parFamily from the pWR100 Virulence Plasmid ofShigella flexneri
Authors: Sergueev Kirill , Dabrazhynetskaya Alena , Austin Stuart
Published: May 2005

P1parfamily members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by aparSpartition site. The large virulence plasmid pWR100 ofShigella flexnericontains a new P1parfamily member: pWR100par. Although typicalparAandparBgenes are present, the putative pWR100parSsite is atypical in sequence and organization. However, pWR100parSpromoted accurate plasmid partition inEscherichia coliwhen the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs withinparSdefine species specificities among previously described family members. Although substantially different from P1parSfrom the P1 plasmid prophage ofE. coli, pWR100parShas the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parSand P1parSis the presence of a 21-bp insert at the center of the pWR100parSsite. Deletion of this insert left much of theparSactivity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.
A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar ofHelicobacter pyloriLipopolysaccharide
Authors: Stead Christopher , Tran An , Ferguson Donald , McGrath Sara , Cotter Robert , Trent Stephen
Published: May 2005

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo.Helicobacter pyloriLPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate thatH. pyloriWaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes ofH. pyloricapable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.
The RssAB Two-Component Signal Transduction System in
Serratia marcescens
Regulates Swarming Motility and Cell Envelope Architecture in Response to Exogenous Saturated Fatty Acids
Authors: Lai Hsin-Chih , Soo Po-Chi , Wei Jun-Rong , Yi Wen-Ching , Liaw Shwu-Jen , Horng Yu-Tze , Lin Shi-Ming , Ho Shen-Wu , Swift Simon , Williams Paul
Published: May 2005

Serratia marcescens
swarms at 30°C but not at 37°C on a nutrient-rich (LB) agar surface. Mini-Tn
5
mutagenesis of
S. marcescens
CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37°C but also earlier and faster than the parent strain swarms at 30°C. Analysis of this mutant revealed that the transposon was inserted into a gene (
rssA
) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (
rssB
) was located.
rssA
and
rssB
insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37°C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30°C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate
S. marcescens
swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the
rssA
and
rssB
mutants grown at 30°C or 37°C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in
Proteus mirabilis
, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.
CrgA Is an Inducible LysR-Type Regulator of
Neisseria meningitidis
, Acting both as a Repressor and as an Activator of Gene Transcription
Authors: Ieva Raffaele , Alaimo Cristina , Delany Isabel , Spohn Gunther , Rappuoli Rino , Scarlato Vincenzo
Published: May 2005

The
crgA
gene of
Neisseria meningitidis
, which codes for a LysR-type regulator, is divergently oriented with respect to the
mdaB
gene, which codes for a hypothetical NADPH-quinone oxidoreductase. Transcriptional studies of the intergenic region between
crgA
and
mdaB
showed that two overlapping and divergent promoters, P

crgA

and P

mdaB

, control transcription of these genes. Deletion of the
crgA
gene led to a strong increase in transcription from the P

crgA

promoter and a concomitant strong decrease in transcription from the P

mdaB

promoter, indicating that CrgA acts both as an autorepressor of transcription at its own promoter and as an activator of transcription at the
mdaB
promoter. Addition of α-methylene-γ-butyrolactone (MBL), an inducer of NADPH-quinone oxidoreductase, to wild-type
N. meningitidis
cells specifically resulted in further activation of transcription of the P

mdaB

promoter and more repression of transcription of the P

crgA

promoter. No such regulation was observed when MBL was added to
crgA
-deficient cells, indicating that the transcriptional response to MBL is CrgA mediated. Under the same experimental conditions, no regulation of transcription by either CrgA or MBL was detected at the pilus and capsule genes. The role of CrgA in the regulation of gene expression during the infectious cycle of
N. meningitidis
is discussed.
Role of
mutS
and
mutL
Genes in Hypermutability and Recombination in
Staphylococcus aureus
Authors: Prunier Anne-Laure , Leclercq Roland
Published: May 2005

The mutator phenotype has been linked in several bacterial genera to a defect in the methyl-mismatch repair system, in which the major components are MutS and MutL. This system is involved both in mismatch repair and in prevention of recombination between homeologous fragments in
Escherichia coli
and has been shown to play an important role in the adaptation of bacterial populations in changing and stressful environments. In this report we describe the molecular analysis of the
mutS
and
mutL
genes of
Staphylococcus aureus
. A genetic analysis of the
mutSL
region was performed in
S. aureus
RN4220. Reverse transcriptase PCR experiments confirmed the operon structure already reported in other gram-positive organisms. Insertional inactivation of
mutS
and
mutL
genes and complementation showed the role of both genes in hypermutability in this species. We also designed an in vitro model to study the role of MutS and MutL in homeologous recombination in
S. aureus
. For this purpose, we constructed a bank of
S. aureus
RN4220 and
mutS
and
mutL
mutants containing the integrative thermosensitive vector pBT1 in which fragments with various levels of identity (74% to 100%) to the
S. aureus sodA
gene were cloned. MutS and MutL proteins seemed to have a limited effect on the control of homeologous recombination. Sequence of
mutS
and
mutL
genes was analyzed in 11 hypermutable
S. aureus
clinical isolates. In four of five isolates with mutated or deleted
mutS
or
mutL
genes, a relationship between alterations and mutator phenotypes could be established by negative complementation of the
mutS
or
mutL
mutants.
YojI ofEscherichia coliFunctions as a Microcin J25 Efflux Pump
Authors: Delgado Mónica A. , Vincent Paula A. , Farías Ricardo N. , Salomón Raúl A.
Published: May 2005

In the present study, we showed thatyojI, anEscherichia coliopen reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy ofyojIincreased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.
Agrobacterium tumefaciensVirB9, an Outer-Membrane-Associated Component of a Type IV Secretion System, Regulates Substrate Selection and T-Pilus Biogenesis
Authors: Jakubowski Simon J. , Cascales Eric , Krishnamoorthy Vidhya , Christie Peter J.
Published: May 2005

Agrobacterium tumefacienstranslocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra−), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+strains failed to elaborate T pilus at detectable levels (Pil−), and three such Tra+Pil−mutant strains were rendered Tra−upon deletion ofvirB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.
Identification of a 521-Kilodalton Protein (Gli521) Involved in Force Generation or Force Transmission for
Mycoplasma mobile
Gliding
Authors: Seto Shintaro , Uenoyama Atsuko , Miyata Makoto
Published: May 2005

Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of
Mycoplasma mobile
, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the
gli521
gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the “neck,” as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.
Structural and Functional Divergence of MutS2 from Bacterial MutS1 and Eukaryotic MSH4-MSH5 Homologs
Authors: Kang Josephine , Huang Shuyan , Blaser Martin J.
Published: May 2005

MutS homologs, identified in nearly all bacteria and eukaryotes, include the bacterial proteins MutS1 and MutS2 and the eukaryotic MutS homologs 1 to 7, and they often are involved in recognition and repair of mismatched bases and small insertion/deletions, thereby limiting illegitimate recombination and spontaneous mutation. To explore the relationship of MutS2 to other MutS homologs, we examined conserved protein domains. Fundamental differences in structure between MutS2 and other MutS homologs suggest that MutS1 and MutS2 diverged early during evolution, with all eukaryotic homologs arising from a MutS1 ancestor. Data from MutS1 crystal structures, biochemical results from MutS2 analyses, and our phylogenetic studies suggest that MutS2 has functions distinct from other members of the MutS family. AmutS2mutant was constructed inHelicobacter pylori, which lacksmutS1and mismatch repair genesmutLandmutH. We show that MutS2 plays no role in mismatch or recombinational repair or deletion between direct DNA repeats. In contrast, MutS2 plays a significant role in limiting intergenomic recombination across a range of donor DNA tested. This phenotypic analysis is consistent with the phylogenetic and biochemical data suggesting that MutS1 and MutS2 have divergent functions.
Comparative Analysis of Differentially Expressed Genes in
Shewanella oneidensis
MR-1 following Exposure to UVC, UVB, and UVA Radiation
Authors: Qiu Xiaoyun , Sundin George W. , Wu Liyou , Zhou Jizhong , Tiedje James M.
Published: May 2005

We previously reported that
Shewanella oneidensis
MR-1 is highly sensitive to UVC (254 nm), UVB (290 to 320 nm), and UVA (320 to 400 nm). Here we delineated the cellular response of MR-1 to UV radiation damage by analyzing the transcriptional profile during a 1-h recovering period after UVC, UVB, and UVA exposure at a dose that yields about a 20% survival rate. Although the SOS response was observed with all three treatments, the induction was more robust in response to short-wavelength UV radiation (UVB and UVC). Similarly, more prophage-related genes were induced by short-wavelength UV radiation. MR-1 showed an active detoxification mechanism in response to UVA, which included the induction of antioxidant enzymes and iron-sequestering proteins to scavenge reactive oxygen species. In addition, a great number of genes encoding multidrug and heavy metal efflux pumps were induced following UVA irradiation. Our data suggested that activation of prophages appears the major lethal factor in MR-1 following UVC or UVB irradiation, whereas oxidative damage contributes greatly to the high UVA sensitivity in MR-1.
Developmental-Stage-Specific Assembly of ParB Complexes inStreptomyces coelicolorHyphae
Authors: Jakimowicz Dagmara , Gust Bertolt , Zakrzewska-Czerwinska Jolanta , Chater Keith F.
Published: May 2005

InStreptomyces coelicolorParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci inS. coelicolorvegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.
Bacteriophage Mu Targets the Trinucleotide Sequence CGG
Authors: Manna Dipankar , Deng Shuang , Breier Adam M. , Higgins N. Patrick
Published: May 2005

Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.
Transcriptome Analysis of the Progressive Adaptation of
Lactococcus lactis
to Carbon Starvation
Authors: Redon Emma , Loubiere Pascal , Cocaign-Bousquet Muriel
Published: May 2005

Adaptation of
Lactococcus lactis
towards progressive carbon starvation is mediated by three different types of transcriptomic responses: (i) global responses, i.e., general decreases of functions linked to bacterial growth and lack of induction of the general stress response; (ii) specific responses functionally related to glucose exhaustion, i.e., underexpression of central metabolism genes, induction of alternative sugar transport and metabolism, and induction of the arginine deiminase pathway; and (iii) other responses never described previously during carbon starvation.
The foliar fungal endophytes of Citrus limon in Argentina
Authors: Durán Estela L , Ploper L Daniel , Ramallo Juan C , Piccolo Grandi Rosely A , Hupper Giancoli Ágata C , Azevedo João L
Published: May 2005

Fungal endophytes associated with Citrus limon (L.) Burm., were investigated. Culture of surface-sterilized leaves from two orchards of lemon trees in Tucumán, located in northwestern Argentina, revealed the presence of endophytic fungi in all leaves examined. The colonization frequencies were 69.7% and 72.3% at the two sampled sites, and a total of nine taxa were isolated. Eight of the taxa had relative frequencies of >2% in at least one sample. The number of isolates was significantly lower in the spring. The assemblage composition at each site and sampling time was similar. Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz., responsible for anthracnose, was consistently dominant. Another pathogenic fungus of Citrus spp., Guignardia citricarpa Kiely was also recovered as an endophyte. Scanning electron micrographs of cut uncultured and cultured leaf surfaces, seen in transverse section, showed intercellular fungal hyphae in parenchyma cells No penetration of host cells by either fungus was observed. This is the first report on fungal endophytes in leaves of lemon plants in Argentina.Key words: endophytic fungi, lemon, colonization, scanning electron microscopy.
Root environmental effects on phi thickening production and root morphology in three gymnosperms
Authors: Gerrath Jean M , Matthes Uta , Purich Melissa , Larson Douglas W
Published: May 2005

Phi thickenings, lignified thickenings found on the radial and tangential walls of root cortical cells, are present in a restricted number of angiosperms and gymnosperms. To test whether their occurrence is constitutive or environmentally induced, seedlings of Cryptomeria japonica (L.f.) D. Don (Cupressaceae, phi thickenings present), and Pinus aristata Engel. and Pinus rigida Mill. (Pinaceae, phi thickenings absent) were grown under conditions of waterlogging and soil compaction. After 4 months, five morphological characters and the presence and cross-sectional area of phi thickenings were measured. All measured aspects of seedling morphology were affected by the treatments, and the two treatment factors (waterlogging and compaction) interacted significantly with one another. Taproots were significantly shorter, lateral roots were shorter and fewer in number, and root:shoot ratios were smaller in waterlogged and compacted soil in all three species. Despite these morphological effects, phi thickening presence or absence did not vary: phi thickenings were always found in C. japonica and never in the two Pinus species. However, the phi ratio (the area occupied by phi thickenings relative to the total root area) decreased significantly with both waterlogging and compaction even though it remained unchanged with root maturity. Thus, although root morphology and phi thickening anatomy were significantly affected by the environment, the presence of phi thickenings was not, and is evidently a constitutive anatomical feature in the species examined.Key words: phi thickenings, root anatomy, Cryptomeria, Pinus, waterlogging, soil compaction.
Molecular markers indicate that the narrow Québec endemicsRosa rousseauiorumandRosa williamsiiare synonymous with the widespreadRosa blanda
Authors: Bruneau Anne , Joly Simon , Starr Julian R , Drouin Josée-Nadia
Published: May 2005

Rosa rousseauiorum Boivin and Rosa williamsii Fern. are two rare roses in eastern Québec, whose taxonomic status is controversial. Morphological characters alone do not clearly differentiate these two taxa from each other or from the morphologically variable and widespread Rosa blanda Ait. We evaluated the taxonomic status of these two taxa, and of two other R. blanda segregates, Rosa subblanda Rydb. and Rosa johannensis Fern., through an analysis of RAPD, ISSR, and AFLP markers. We surveyed 86 individuals from 36 populations in eastern North America. Despite a high degree of polymorphism, principal coordinate analyses and the weighted pair group method with arithmetic averaging suggest no clustering of individuals that correspond to taxonomic boundaries. However, the closely related Rosa palustris Marsh. is clearly differentiated from the R. blanda s.l. taxa. When populations of R. blanda west of Québec are included, the principal coordinate analyses and Mantel tests indicate the presence of a significant eastwest geographic gradient. Analyses of molecular variation suggest that most of the observed variation occurs within taxa, rather than among taxa. A weak inter-taxon variation is nonetheless significant for RAPD and ISSR data, and a weak pattern dependent on geographical location is evident within the province of Québec. In accordance with studies based on morphological characters, molecular data indicate that R. rousseauiorum and R. williamsii should not be considered as species distinct from R. blanda.Key words: Rosa blanda, Rosa rousseauiorum, Rosa williamsii, Rosa johannensis, Rosa subblanda, RAPD, ISSR, AFLP, endangered plants, taxonomic status.
Residual effects of NPK fertilization on shrub growth in a Yukon boreal forest
Authors: Melnychuk Michael C , Krebs Charles J
Published: May 2005

Residual effects of nutrient additions were sustained in shrubs 48 years after fertilization stopped in a northern boreal forest in Yukon, Canada. We measured the growth rate of grey willow (Salix glauca L.) and bog birch (Betula glandulosa Michx.) twigs during the growing seasons of 1998, 2001, and 2002, 48 years after NPK fertilization from 1987 to 1994 had ceased. We also measured the nitrogen concentration of the 1998 growth tissue. Willow twigs had significantly higher growth rates in previously fertilized shrubs than control shrubs in these 3 years, even greater than differences observed during fertilization. Willow also had higher N concentration in fertilized twigs than control twigs. Birch growth was negatively correlated with hare abundance during the snowshoe hare (Lepus americanus) abundance cycle. Birch growth rate was higher in fertilized twigs than control twigs in 1998 during the hare peak, but fertilized birch twigs approached control levels by 2002 following the hare decline.Key words: Salix, Betula, productivity, nitrogen, fertilizer, herbivory.
Analysis of the spatial genetic structure ofPassiflora incarnatain recently disturbed sites
Authors: Tague Rebecca T , Foré Stephanie A
Published: May 2005

In early successional species, short life span and frequent spatial relocation may affect the distribution of genetic variation but the pattern may be altered by reproductive patterns. Passiflora incarnata L. (Passifloraceae), an early successional vine found throughout the southeastern United States, reproduces sexually and asexually through clonal sprouts. We examined the genetic structure of P. incarnata in recently disturbed habitats at three spatial scales: within a patch, among patches separated by 250 m, and between sites separated by 10 km. Genetic variation may be clumped at the scale of neighboring plants if stem resprouting is significant. In each patch, eleven arbitrarily selected plants and their four nearest neighbors were mapped and leaf samples were collected for genetic analysis. The multilocus genotype of each individual for seven polymorphic allozymes was determined. Potential clones were determined by estimating the probability of a second occurrence of each genotype and a multilocus coefficient of coancestry. Data indicated P. incarnata was reproducing primarily sexually. Most of the genetic variation was within a patch with little variation among patches. These data suggest that the genetic structure of this colonizing species was determined by founder effects interacting with long distance pollen movement.Key words: allozymes, passionflower, spatial, genetic structure, early colonizer, Passiflora incarnata.
The effect of gut passage by two species of avian frugivore on seeds of pokeweed, Phytolacca americana
Authors: Orrock John L
Published: May 2005

Although avian frugivores are known to be important dispersers of seeds of pokeweed, Phytolacca americana L., there are no studies that rigorously examine the effect of gut passage through avian frugivores on P. americana seeds. I examined how passage through avian frugivores affected the proportion of P. americana seeds germinating, the rate of germination (average number of days required for all seeds to germinate), and the total number of viable seeds. Field-collected fruits were either cleaned of pulp (control seeds), fed to northern Mockingbirds (Mimus polyglottos), or fed to Brown Thrashers (Toxostoma rufum). The proportion of seeds germinating after passage through avian frugivores was greater than control seeds (0.88 vs. 0.67, respectively), but did not differ between Mockingbirds or Brown Thrashers. However, seeds consumed by Mockingbirds germinated significantly faster on average (4.2 d) compared with seeds consumed by Brown Thrashers (4.6 d). Consumption by either species led to faster germination than control seeds (5.5 d). The total number of viable seeds did not differ among seeds consumed by avian frugivores or control seeds. These results suggest that avian frugivores do not change the viability of P. americana seeds. Rather, avian frugivores shifted the timing of germination, such that more seeds germinate more quickly after passage through frugivores. The adaptive implications of accelerated germination following passage through frugivores are briefly discussed.Key words: dispersal, frugivore, germination, viability.