bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectral peak resolution and speech recognition in quiet: Normal hearing, hearing impaired, and cochlear implant listeners
Authors: Henry Belinda A. , Turner Christopher W. , Behrens Amy
Published: August 2005

Spectral peak resolution was investigated in normal hearing (NH), hearing impaired (HI), and cochlear implant (CI) listeners. The task involved discriminating between two rippled noise stimuli in which the frequency positions of the log-spaced peaks and valleys were interchanged. The ripple spacing was varied adaptively from 0.13 to 11.31 ripples/octave, and the minimum ripple spacing at which a reversal in peak and trough positions could be detected was determined as the spectral peak resolution threshold for each listener. Spectral peak resolution was best, on average, in NH listeners, poorest in CI listeners, and intermediate for HI listeners. There was a significant relationship between spectral peak resolution and both vowel and consonant recognition in quiet across the three listener groups. The results indicate that the degree of spectral peak resolution required for accurate vowel and consonant recognition in quiet backgrounds is around 4 ripples/octave, and that spectral peak resolution poorer than around 1–2 ripples/octave may result in highly degraded speech recognition. These results suggest that efforts to improve spectral peak resolution for HI and CI users may lead to improved speech recognition.
Analysis and synthesis of the three-dimensional movements of the head, face, and hand of a speaker using cued speech
Authors: Gibert Guillaume , Bailly Gérard , Beautemps Denis , Elisei Frédéric , Brun Rémi
Published: August 2005

In this paper we present efforts for characterizing the three dimensional (3-D) movements of the right hand and the face of a French female speaker during the audiovisual production of cued speech. The 3-D trajectories of 50 hand and 63 facial flesh points during the production of 238 utterances were analyzed. These utterances were carefully designed to cover all possible diphones of the French language. Linear and nonlinear statistical models of the articulations and the postures of the hand and the face have been developed using separate and joint corpora. Automatic recognition of hand and face postures at targets was performed to verify a posteriori that key hand movements and postures imposed by cued speech had been well realized by the subject. Recognition results were further exploited in order to study the phonetic structure of cued speech, notably the phasing relations between hand gestures and sound production. The hand and face gestural scores are studied in reference with the acoustic segmentation. A first implementation of a concatenative audiovisual text-to-cued speech synthesis system is finally described that employs this unique and extensive data on cued speech in action.
Optical and tomographic imaging of a middle ear malformation in the bullfrog (Rana catesbeiana)
Authors: Horowitz Seth S. , Simmons Andrea Megela , Ketten Darlene R.
Published: August 2005

Using a combination of in vivo computerized tomography and histological staining, a middle ear anomaly in two wild-caught American bullfrogs (Rana catesbeiana) is characterized. In these animals, the tympanic membrane, extrastapes, and pars media (shaft) of the stapes are absent on one side of the head, with the other side exhibiting normal morphology. The pars interna (footplate) of the stapes and the operculum are present in their normal positions at the entrance of the otic capsule on both the affected and unaffected sides. The pattern of deformity suggests a partial failure of development of tympanic pathway tissues, but with a preservation of the opercularis pathway. While a definitive proximate cause of the condition could not be determined, the anomalies show similarities to developmental defects in mammalian middle ear formation.
On the relative importance of spatial and timbral fidelities in judgments of degraded multichannel audio quality
Authors: Rumsey Francis , Zieliński Sławomir , Kassier Rafael , Bech Søren
Published: August 2005

Mean opinion score ratings of reproduced sound quality typically pool all contributing perceptual factors into a single rating of basic audio quality. In order to improve understanding of the trade-offs between selected sound quality degradations that might arise in systems for the delivery of high quality multichannel audio, it was necessary to evaluate the influence of timbral and spatial fidelity changes on basic audio quality grades. The relationship between listener ratings of degraded multichannel audio quality on one timbral and two spatial fidelity scales was exploited to predict basic audio quality ratings of the same material using a regression model. It was found that timbral fidelity ratings dominated but that spatial fidelity predicted a substantial proportion of the basic audio quality.
Wavelet preprocessing for lessening truncation effects in nearfield acoustical holography
Authors: Thomas Jean-Hugh , Pascal Jean-Claude
Published: August 2005

The goal of planar nearfield acoustical holography (NAH) is to recover the sound field at the sound source from pressure measurements made close to the source plane. The theory requires the pressure to be measured over a complete plane. Because experimentation consists of acquiring only a finite measurement aperture of the pressure field, it naturally causes erroneous values in the reconstructed field. Wavelet preprocessing applied to the pressure measurements in the nearfield provides a solution to lessen the effects due to the truncation of the hologram. The approach is based on a multiresolution analysis of the field from different wave number bands followed by selective spatial filtering of effects highlighted by the first analysis. Experimental results show the relevance of the method by comparison to standard NAH involving exponential filtering in the wave number domain. The computation of objective indicators based on distance measurements between wave number spectra and comparisons between patterns composed of relevant features drawn from experimental data are proposed to give objective criteria to prove the viability of the method.
Can dichotic pitches form two streams?
Authors: Akeroyd Michael A. , Carlyon Robert P. , Deeks John M.
Published: August 2005

The phenomenon of auditory streaming reflects the perceptual organization of sounds over time. A series of “A” and “B” tones, presented in a repeating “ABA-ABA” sequence, may be perceived as one “galloping” stream or as two separate streams, depending on the presentation rate and the A-B frequency separation. The present experiment examined whether streaming occurs for sequences of “Huggins pitches,” for which the percepts of pitch are derived from the binaural processing of a sharp transition in interaural phase in an otherwise diotic noise. Ten-second “ABA” sequences were presented to eight normal-hearing listeners for two types of stimuli: Huggins-pitch stimuli with interaural phase transitions centered on frequencies between 400 and 800Hz, or partially-masked diotic tones-in-noise, acting as controls. Listeners indicated, throughout the sequence, the number of streams perceived. The results showed that, for both Huggins-pitch stimuli and tones-in-noise, two streams were often reported. In both cases, the amount of streaming built up over time, and depended on the frequency separation between the A and B tones. These results provide evidence that streaming can occur between stimuli whose pitch percept is derived binaurally. They are inconsistent with models of streaming based solely on differences in the monaural excitation pattern.
Multiresolution spectrotemporal analysis of complex sounds
Authors: Chi Taishih , Ru Powen , Shamma Shihab A.
Published: August 2005

A computational model of auditory analysis is described that is inspired by psychoacoustical and neurophysiological findings in early and central stages of the auditory system. The model provides a unified multiresolution representation of the spectral and temporal features likely critical in the perception of sound. Simplified, more specifically tailored versions of this model have already been validated by successful application in the assessment of speech intelligibility [Elhilali et al., Speech Commun. 41(2-3), 331–348 (2003); Chi et al., J. Acoust. Soc. Am. 106, 2719–2732 (1999)] and in explaining the perception of monaural phase sensitivity [R. Carlyon and S. Shamma, J. Acoust. Soc. Am. 114, 333–348 (2003)]. Here we provide a more complete mathematical formulation of the model, illustrating how complex signals are transformed through various stages of the model, and relating it to comparable existing models of auditory processing. Furthermore, we outline several reconstruction algorithms to resynthesize the sound from the model output so as to evaluate the fidelity of the representation and contribution of different features and cues to the sound percept.
Field impact insulation testing: Inadequacy of existing normalization methods and proposal for new ratings analogous to those for airborne noise reduction
Authors: LoVerde John J. , Dong D. Wayland
Published: August 2005

The field test method for determining the Field Impact Isolation Class (FIIC) rating of a floor/ceiling assembly, prescribed in ASTM E 1007, requires an estimation of the receiving room absorption and the normalization of the receiving room sound pressure levels based on a standard quantity of room absorption. Normalization is intended to remove the effects of receiving room acoustical characteristics, but an analysis of field impact testing indicates that this method has a limited engineering value. The normalization correction is strongly dependent on receiving room volume, does not correlate with the normalization used in airborne sound isolation testing, and can be unreasonably large under certain conditions. The current test method leads to potential errors in engineering judgment, as illustrated by example field tests. By contrast, airborne noise reduction ratings (prescribed in ASTM E 336) are either non-normalized or normalized to a standard reverberation time, which provides significant advantages over normalizing to a standard amount of absorption. New impact noise metrics, the non-normalized impact sound rating (ISR) and the normalized impact sound rating (NISR), analogous to the airborne noise reduction metrics NIC and NNIC, are proposed for incorporation into ASTM E 1007. Revised building code requirements using the proposed metrics are suggested.
The energy method for analyzing the piezoelectric electroacoustic transducers. II. (With the examples of the flexural plate transducer)
Authors: Aronov Boris
Published: August 2005

The energy method for analyzing piezoelectric electro-acoustic transducers, described in the previous paper [B. S. Aronov, J. Acoust. Soc. Am. 117, 210–220 (2005)] is used here in application to transducers operating in the receive mode. Changes to the equivalent circuit of a transducer are introduced, arising from the action of the sound field considered as the external source of energy. The application of the energy method is demonstrated by an example of the circular flexural plate transducer. The validity of the single degree of freedom approximation for calculating the transducer parameters is considered, and estimation is made of inaccuracies arising from the simplifying assumptions regarding the mode of transducer vibration. The acceptable tolerance levels for accuracy in the calculation of the transducer parameters are also discussed.
Supplementary notes on the Gaussian beam expansion
Authors: Ding Desheng , Tong Xiangjie , He Peizhong
Published: August 2005

The letter provides alternatively a simple way of computing the Fresnel field integral, a further extension to the Gaussian-beam expansion. The zeroth-order Bessel function of the first kind is expanded into an approximate sum of Gaussian functions. The field integral is then expressible in terms of these simple functions. The approach is useful in treatment of the field radiation problem for a large and important group of piston sources in acoustics. As examples, the calculation results for the uniform and the simply-supported piston sources are presented, in a good agreement with those evaluated by numerical integration.
Predicting fundamental frequency from mel-frequency cepstral coefficients to enable speech reconstruction
Authors: Shao Xu , Milner Ben
Published: August 2005

This work proposes a method to reconstruct an acoustic speech signal solely from a stream of mel-frequency cepstral coefficients (MFCCs) as may be encountered in a distributed speech recognition (DSR) system. Previous methods for speech reconstruction have required, in addition to the MFCC vectors, fundamental frequency and voicing components. In this work the voicing classification and fundamental frequency are predicted from the MFCC vectors themselves using two maximum a posteriori (MAP) methods. The first method enables fundamental frequency prediction by modeling the joint density of MFCCs and fundamental frequency using a single Gaussian mixture model (GMM). The second scheme uses a set of hidden Markov models (HMMs) to link together a set of state-dependent GMMs, which enables a more localized modeling of the joint density of MFCCs and fundamental frequency. Experimental results on speaker-independent male and female speech show that accurate voicing classification and fundamental frequency prediction is attained when compared to hand-corrected reference fundamental frequency measurements. The use of the predicted fundamental frequency and voicing for speech reconstruction is shown to give very similar speech quality to that obtained using the reference fundamental frequency and voicing.
Acoustic-phonetics of coronal stops: A cross-language study of Canadian English and Canadian French
Authors: Sundara Megha
Published: August 2005

The study was conducted to provide an acoustic description of coronal stops in Canadian English (CE) and Canadian French (CF). CE and CF stops differ in VOT and place of articulation. CE has a two-way voicing distinction (in syllable initial position) between simultaneous and aspirated release; coronal stops are articulated at alveolar place. CF, on the other hand, has a two-way voicing distinction between prevoiced and simultaneous release; coronal stops are articulated at dental place. Acoustic analyses of stop consonants produced by monolingual speakers of CE and of CF, for both VOT and alveolar/dental place of articulation, are reported. Results from the analysis of VOT replicate and confirm differences in phonetic implementation of VOT across the two languages. Analysis of coronal stops with respect to place differences indicates systematic differences across the two languages in relative burst intensity and measures of burst spectral shape, specifically mean frequency, standard deviation, and kurtosis. The majority of CE and CF talkers reliably and consistently produced tokens differing in the SD of burst frequency, a measure of the diffuseness of the burst. Results from the study are interpreted in the context of acoustic and articulatory data on coronal stops from several other languages.
Acoustics of the human middle-ear air space
Authors: Stepp Cara E. , Voss Susan E.
Published: August 2005

The impedance of the middle-ear air space was measured on three human cadaver ears with complete mastoid air-cell systems. Below 500 Hz, the impedance is approximately compliance-like, and at higher frequencies (500–6000 Hz) the impedance magnitude has several (five to nine) extrema. Mechanisms for these extrema are identified and described through circuit models of the middle-ear air space. The measurements demonstrate that the middle-ear air space impedance can affect the middle-ear impedance at the tympanic membrane by as much as 10 dB at frequencies greater than 1000 Hz. Thus, variations in the middle-ear air space impedance that result from variations in anatomy of the middle-ear air space can contribute to inter-ear variations in both impedance measurements and otoacoustic emissions, when measured at the tympanic membrane.
Sensitivity to changing stimulus distribution characteristics in auditory categorization
Authors: Sullivan Sarah C. , Lotto Andrew J. , Newlin Elizabeth T. , Diehl Randy L.
Published: August 2005

This experiment examined the ability of humans to categorize sounds as a function of changing training distribution characteristics. Participants were presented non-speech sounds randomly sampled from two overlapping distributions. The sounds consisted of 25 narrow-band noise bursts varying in center frequency from 1000 to 1360 Hz. Four different distribution pairs were created by varying the ratio of stimuli in each distribution (i.e., prior probabilities), resulting in different distribution cross-over points or ideal boundaries. Subjects identified the distribution from which each sound was drawn and were provided feedback. The training distributions were switched midsession without alerting the subjects. Performance was tracked by plotting identification functions and noting boundary placement for each individual block. Previously collected data suggested that the ability to learn optimal boundaries was determined in part by the proximity of the distribution boundary to the stimulus midrange point. Results from the current experiment also suggest that distance from the midrange point constrains learning. Subjects were able to learn optimal or near-optimal boundary placement for overlapping distributions only when the ideal boundary was near the midrange point and when those distributions were presented during the initial blocks of training in the experiment. [Work supported by NSF and NIDCD.]
Practical aspects of in situ directional measurements
Authors: Saltykov Oleg
Published: August 2005

This paper describes practical aspects of measuring in situ directionality in simulated diffuse field. The method is based on exposing the object to a sequence of acoustic stimuli coming from a number of sound sources located on a surface of a test sphere. Empirical data are presented that describe how the number of elements in the spherical array affects the measurement errors. Construction of a spherical array is described that simplifies the test procedure and assures measurement errors less than 0.1 dB. A test setup for fast automated measurements of in situ directionality has been developed. The time delay spectrometry (TDS) method has been chosen for directional measurements because of its ability to improve signal-to-noise ratio. Another big advantage of the TDS method is its ability to filter out some undesirable acoustic reflections, therefore improving anechoic conditions in the test space. It should be possible to conduct in situ directional tests in a large, nonanechoic room with the TDS method.
Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome
Authors: Shendure Jay , Porreca Gregory J. , Reppas Nikos B. , Lin Xiaoxia , McCutcheon John P. , Rosenbaum Abraham M. , Wang Michael D. , Zhang Kun , Mitra Robi D. , Church George M.
Published: August 2005

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of
Escherichia coli
at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.
Comparison between Cultured Small-Intestinal and Fecal Microbiotas in Beagle Dogs
Authors: Mentula Silja , Harmoinen Jaana , Heikkilä Matti , Westermarck Elias , Rautio Merja , Huovinen Pentti , Könönen Eija
Published: August 2005

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10
2
to 10
6
CFU/g) than in feces (range, 10
8
to 10
11
CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant
Bacteroides
spp.,
Clostridium hiranonis
-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.
Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium,
Lactobacillus thermotolerans
, in Chicken Feces
Authors: Selim Abu Sadeque Md , Boonkumklao Piyanuch , Sone Teruo , Assavanig Apinya , Wada Masaru , Yokota Atsushi
Published: August 2005

A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium,
Lactobacillus thermotolerans
, in chicken feces. The specific primers and probe were designed based on the
L. thermotolerans
16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of
L. thermotolerans
, DNA from the closely related bacteria
Lactobacillus mucosae
DSM 13345
T
and
Lactobacillus fermentum
JCM 1173
T
, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of
L. thermotolerans
DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 10
3
cells/ml of an
L. thermotolerans
pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10
4
cells/g feces on day 4 and 10
5
cells/g feces on days 11 to 18. However, cell populations of 10
6
to 10
7
cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 10
11
cells/g feces. These results suggest that in general,
L. thermotolerans
is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.
Bacteriophages May Bias Outcome of Bacterial Enrichment Cultures
Authors: Muniesa Maite , Blanch Anicet R. , Lucena Francisco , Jofre Juan
Published: August 2005

Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages inSalmonellaandShigellaenrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-μm non-protein-binding membranes. Furthermore, our data indicate that theSalmonellabiotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis.
Effects of Bacteria on
Artemia franciscana
Cultured in Different Gnotobiotic Environments
Authors: Marques Antonio , Dinh Thi , Ioakeimidis Christos , Huys Geert , Swings Jean , Verstraete Willy , Dhont Jean , Sorgeloos Patrick , Bossier Peter
Published: August 2005

The use of probiotics is receiving considerable attention as an alternative approach to control microbiota in aquaculture farms, especially in hatching facilities. However, application with consistent results is hampered by insufficient information on their modes of action. To investigate whether dead bacteria (allowing investigation of their nutritional effect) or live bacteria (allowing evaluation of their probiotic effect) have any beneficial effect towards
Artemia franciscana
and, subsequently, if live bacteria have probiotic effects beyond the effects observed with dead bacteria, a model system was employed using gnotobiotic
Artemia
as a test organism. Nauplii were cultured in the presence of 10 bacterial strains combined with four different major axenic live feeds (two strains of
Saccharomyces cerevisiae
and two strains of
Dunaliella tertiolecta
) differing in their nutritional values. In combination with poor- and medium-quality live feeds, dead bacteria exerted a strong effect on
Artemia
survival but a rather weak or no effect on individual length and constituted a maximum of only 5.9% of the total ash-free dry weight supplied. These effects were reduced or even disappeared when medium- to good-quality major feed sources were used, possibly due to improvements in the health status of
Artemia
. Some probiotic bacteria, such as GR 8 (
Cytophaga
spp.), improved (not always significantly) the performance of nauplii beyond the effect observed with dead bacteria, independently of the feed supplied. The present approach can be an excellent system to study the exact mode of action of bacteria, especially if combined with challenge tests or other types of analysis (e.g., transcriptome and proteonomic analysis).
Phylogenetic Characterization of a Polychlorinated-Dioxin- Dechlorinating Microbial Community by Use of Microcosm Studies
Authors: Yoshida Naoko , Takahashi Nobutaka , Hiraishi Akira
Published: August 2005

Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phylaFirmicutes,Proteobacteria, andBacteroidetespredominated. A significant number ofChloroflexiclones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, “Dehalococcoides,” and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical “Dehalococcoides” group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the “Dehalococcoides”-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.
Phosphate Starvation-Inducible Gene
ushA
Encodes a 5′ Nucleotidase Required for Growth of
Corynebacterium glutamicum
on Media with Nucleotides as the Phosphorus Source
Authors: Rittmann Doris , Sorger-Herrmann Ulrike , Wendisch Volker F.
Published: August 2005

Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5′ nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of
Escherichia coli
, or in phosphorus metabolism. The
Corynebacterium glutamicum
gene
ushA
was found to encode a secreted enzyme which is active as a 5′ nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when
C. glutamicum
was starved for inorganic phosphate. UshA was required for growth of
C. glutamicum
on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from
E. coli
,
C. glutamicum
UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus.
Two Rhizobial Strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities
Authors: Sato Yukari , Monincová Marta , Chaloupková Radka , Prokop Zbyňek , Ohtsubo Yoshiyuki , Minamisawa Kiwamu , Tsuda Masataka , Damborský Jiří , Nagata Yuji
Published: August 2005

Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, have open reading frames (ORFs),mlr5434andblr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts ofEscherichia colistrains expressingmlr5434andblr1087showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to asdmlA(dehalogenase fromMesorhizobium loti) anddbjA(dehalogenase fromBradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards β-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more α-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that thedmlAanddbjAgenes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.
Correlation of Phenotype with the Genotype of Egg-Contaminating
Salmonella enterica
Serovar Enteritidis
Authors: Morales Cesar A. , Porwollik Steffen , Frye Jonathan G. , Kinde Hailu , McClelland Michael , Guard-Bouldin Jean
Published: August 2005

The genotype of
Salmonella enterica
serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37°C. However, the wild-type
S. enterica
serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25°C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that
S. enterica
serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25°C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37°C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the
S. enterica
serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.
Expression of the
psl
Operon in
Pseudomonas aeruginosa
PAO1 Biofilms: PslA Performs an Essential Function in Biofilm Formation
Authors: Overhage Jörg , Schemionek Mirle , Webb Jeremy S. , Rehm Bernd H. A.
Published: August 2005

The
psl
gene cluster, comprising 15 cotranscribed genes from
Pseudomonas aeruginosa
, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the
psl
gene cluster and the function of the first gene in this cluster, the
pslA
gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free
pslA
deletion mutant of
P. aeruginosa
PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the
pslA
gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid
P. aeruginosa
. The promoter region of the
psl
gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5′ ends. Promoter assays using transcriptional fusions to
lacZ
and
gfp
indicated a constitutive expression of the
psl
cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the
psl
cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the
psl
operon enables efficient attachment to surfaces and that regulated localized
psl
operon expression is required for biofilm differentiation.
Allophanate Hydrolase, Not Urease, Functions in Bacterial Cyanuric Acid Metabolism
Authors: Cheng Gang , Shapir Nir , Sadowsky Michael J. , Wackett Lawrence P.
Published: August 2005

Growth substrates containing an
s
-triazine ring are typically metabolized by bacteria to liberate 3 mol of ammonia via the intermediate cyanuric acid. Over a 25-year period, a number of original research papers and reviews have stated that cyanuric acid is metabolized in two steps to the 2-nitrogen intermediate urea. In the present study, allophanate, not urea, was shown to be the 2-nitrogen intermediate in cyanuric acid metabolism in all the bacteria examined. Six different experimental results supported this conclusion: (i) synthetic allophanate was shown to readily decarboxylate to form urea under acidic extraction and chromatography conditions used in previous studies; (ii) alkaline extraction methods were used to stabilize and detect allophanate in bacteria actively metabolizing cyanuric acid; (iii) the kinetic course of allophanate formation and disappearance was consistent with its being an intermediate in cyanuric acid metabolism, and no urea was observed in those experiments; (iv) protein extracts from cells grown on cyanuric acid contained allophanate hydrolase activity; (v) genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze allophanate, respectively, were found in several cyanuric acid-metabolizing bacteria; and (vi) TrzF, an AtzF homolog found in
Enterobacter cloacae
strain 99, was cloned, expressed in
Escherichia coli
, and shown to have allophanate hydrolase activity. In addition, we have observed that there are a large number of genes homologous to
atzF
and
trzF
distributed in phylogenetically distinct bacteria. In total, the data indicate that
s
-triazine metabolism in a broad class of bacteria proceeds through allophanate via allophanate hydrolase, rather than through urea using urease.
Use of
Saccharomyces cerevisiae
BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds
Authors: Sanseverino John , Gupta Rakesh K. , Layton Alice C. , Patterson Stacey S. , Ripp Steven A. , Saidak Leslie , Simpson Michael L. , Schultz T. Wayne , Sayler Gary S.
Published: August 2005

An estrogen-inducible bacterial
lux
-based bioluminescent reporter was developed in
Saccharomyces cerevisiae
for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated
S. cerevisiae
BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express
luxA
and
luxB
to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (
luxCDE
) and FMN reduction (
frp
) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast
lac
Z reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10
−8
through 5.6 × 10
−12
M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (
n
= 7) were similar at (4.4 ± 1.1) × 10
−10
and (2.4 ± 1.0) × 10
−10
M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10
−11
to 2.8 × 10
−9
M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.
Characterization of Botulinum Progenitor Toxins by Mass Spectrometry
Authors: Hines Harry B. , Lebeda Frank , Hale Martha , Brueggemann Ernst E.
Published: August 2005

Botulinum toxin analysis has renewed importance. This study included the use of nanochromatography-nanoelectrospray-mass spectrometry/mass spectrometry to characterize the protein composition of botulinum progenitor toxins and to assign botulinum progenitor toxins to their proper serotype and strain by using currently available sequence information.
Clostridium botulinum
progenitor toxins from strains Hall, Okra, Stockholm, MDPH, Alaska, and Langeland and 89 representing serotypes A through G, respectively, were reduced, alkylated, digested with trypsin, and identified by matching the processed product ion spectra of the tryptic peptides to proteins in accessible databases. All proteins known to be present in progenitor toxins from each serotype were identified. Additional proteins, including flagellins, ORF-X1, and neurotoxin binding protein, not previously reported to be associated with progenitor toxins, were present also in samples from several serotypes. Protein identification was used to assign toxins to a serotype and strain. Serotype assignments were accurate, and strain assignments were best when either sufficient nucleotide or amino acid sequence data were available. Minor difficulties were encountered using neurotoxin-associated protein identification for assigning serotype and strain. This study found that combined nanoscale chromatographic and mass spectrometric techniques can characterize
C. botulinum
progenitor toxin protein composition and that serotype/strain assignments based upon these proteins can provide accurate serotype and, in most instances, strain assignments using currently available information. Assignment accuracy will continue to improve as more nucleotide/amino acid sequence information becomes available for different botulinum strains.
Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium,
Rhodopseudomonas palustris
Strain TIE-1
Authors: Jiao Yongqin , Kappler Andreas , Croal Laura R. , Newman Dianne K.
Published: August 2005

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H
2
, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species
Rhodopseudomonas palustris
, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (α-FeOOH) forms, and above pH 7.2 magnetite (Fe
3
O
4
) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of ∼10
−5
. To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B
12
) biosynthesis.
Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in
Saccharomyces cerevisiae
Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation
Authors: Mensonides Femke I. C. , Brul Stanley , Klis Frans M. , Hellingwerf Klaas J. , Teixeira de Mattos M. Joost
Published: August 2005

This paper reports on physiological and molecular responses of
Saccharomyces cerevisiae
to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a
tps1
mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such.
Involvement of a Branched-Chain Aminotransferase in Production of Volatile Sulfur Compounds in
Yarrowia lipolytica
Authors: Cernat Bondar Daniela , Beckerich Jean-Marie , Bonnarme Pascal
Published: August 2005

The enzymatic degradation of
l
-methionine and the subsequent formation of volatile sulfur compounds (VSCs) are essential for the development of the typical flavor in cheese. In the yeast
Yarrowia lipolytica
, the degradation of
l
-methionine was accompanied by the formation of the transamination product 4-methylthio-2-oxobutyric acid. A branched-chain aminotransferase gene (Yl
BCA1
) of
Y. lipolytica
was amplified, and the
l
-methionine-degrading activity and the aminotransferase activity were measured in a genetically modified strain and compared to those of the parental strain. Our work shows that
l
-methionine degradation via transamination is involved in formation of VSCs in
Y. lipolytica
.
Characterization of an Inducible Phenylserine Aldolase from
Pseudomonas putida
24-1
Authors: Misono Haruo , Maeda Hiroshi , Tuda Kouiti , Ueshima Sakuko , Miyazaki Naoto , Nagata Shinji
Published: August 2005

An inducible phenylserine aldolase (
l
-
threo
-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of
l
-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of
Pseudomonas putida
24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5′ phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on
l
-
threo
-3-phenylserine (

K
m

, 1.3 mM),
l
-
erythro
-3-phenylserine (

K
m

, 4.6 mM),
l
-threonine (

K
m

, 29 mM), and
l
-
allo
-threonine (

K
m

, 22 mM). In the reverse reaction,
threo
- and
erythro
- forms of
l
-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from
Pseudomonas putida
24-1 was cloned and overexpressed in
Escherichia coli
cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5′-phosphate.
Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria
Authors: Naz Naghma , Young Hilary K. , Ahmed Nuzhat , Gadd Geoffrey M.
Published: August 2005

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains
Desulfovibrio desulfuricans
DSM 1926 and
Desulfococcus multivorans
DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the
smtAB
,
cadAC
, and
cadD
genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known
smtAB
genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of
Desulfomicrobium norvegicum
I1 and
Desulfovibrio desulfuricans
DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.