bluuebunny/crossref_metadata_2025 huggingface dataset:
A test of the Equal-Loudness-Ratio hypothesis using cross-modality matching functions
Authors: Epstein Michael , Florentine Mary
Published: August 2005

This study tests the Equal-Loudness-Ratio hypothesis [Florentine et al., J. Acoust. Soc. Am. 99, 1633–1644 (1996)], which states that the loudness ratio between equal-SPL long and short tones is independent of SPL. The amount of temporal integration (i.e., the level difference between equally loud short and long sounds) is maximal at moderate levels. Therefore, the Equal-Loudness-Ratio hypothesis predicts that the loudness function is shallower at moderate levels than at low and high levels. Equal-loudness matches and cross-modality string-length matches were used to assess the form of the loudness function for 5 and 200 ms tones at 1 kHz and the loudness ratio between them. Results from nine normal listeners show that (1) the amount of temporal integration is largest at moderate levels, in agreement with previous studies, and (2) the loudness functions are shallowest at moderate levels. For eight of the nine listeners, the loudness ratio between the 200 and 5 ms tones is approximately constant, except at low levels where it tends to increase. The average data show good agreement between the two methods, but discrepancies are apparent for some individuals. These findings support the Equal-Loudness-Ratio hypothesis, except at low levels.
Time domain computational modeling of viscothermal acoustic propagation in catalytic converter substrates with porous walls
Authors: Dickey N. S. , Selamet A. , Miazgowicz K. D. , Tallio K. V. , Parks S. J.
Published: August 2005

Models for viscothermal effects in catalytic converter substrates are developed for time domain computational methods. The models are suitable for use in one-dimensional approaches for the prediction of exhaust system performance (engine tuning characteristics) and radiated sound levels. Starting with the “low reduced frequency” equations for viscothermal acoustic propagation in capillary tubes, time domain submodels are developed for the frequency-dependent wall friction, frequency-dependent wall heat transfer, and porous wall effects exhibited by catalytic converter substrates. Results from a time domain computational approach employing these submodels are compared to available analytical solutions for the low reduced frequency equations. The computational results are shown to agree well with the analytical solutions for capillary geometries representative of automotive catalytic converter substrates.
Halotolerant Cyanobacterium
Aphanothece halophytica
Contains NapA-Type Na
+
/H
+
Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH
Authors: Wutipraditkul Nuchanat , Waditee Rungaroon , Incharoensakdi Aran , Hibino Takashi , Tanaka Yoshito , Nakamura Tatsunosuke , Shikata Masamitsu , Takabe Tetsuko , Takabe Teruhiro
Published: August 2005

Aphanothece halophytica
is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium
Synechocystis
sp. strain PCC 6803 contains five putative Na
+
/H
+
antiporters, two of which are homologous to NhaP of
Pseudomonas aeruginosa
and three of which are homologous to NapA of
Enterococcus hirae
. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in
Synechocystis
sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in
Aphanothece halophytica
. Two genes encoding polypeptides of the same size, designated Ap-
napA1
-
1
and Ap-
napA1
-
2
, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the
Synechocystis
ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an
Escherichia coli
mutant and exhibited strongly pH-dependent Na
+
/H
+
and Li
+
/H
+
exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K
+
uptake-deficient
E. coli
mutant and exhibited K
+
uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium
Synechococcus
sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that
A. halophytica
has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.
Inhibition of
Clostridium perfringens
by a Novel Strain of
Bacillus subtilis
Isolated from the Gastrointestinal Tracts of Healthy Chickens
Authors: Teo Alex Yeow-Lim , Tan Hai-Meng
Published: August 2005

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against
Clostridium perfringens
, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of
Bacillus subtilis
, was found to possess an anticlostridial factor that could inhibit the
C. perfringens
ATCC 13124 used in this study. The anticlostridial factor produced by
B. subtilis
PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121°C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37°C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of
C. perfringens
implicated in necrotic enteritis in poultry,
Clostridium difficile
,
Streptococcus pneumoniae
,
Campylobacter jejuni
, and
Campylobacter coli.
Use of Enrichment Culture for Directed Evolution of the
Vibrio fluvialis
JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones
Authors: Yun Hyungdon , Hwang Bum-Yeol , Lee Jae-Hun , Kim Byung-Gee
Published: August 2005

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant ω-transaminase from
Vibrio fluvialis
JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, ω-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using ω-TAmla (50 U/ml) overexpressed in
Escherichia coli
BL21, 150 mM 2-aminoheptane was successfully resolved to (
R
)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.
Study of Adhesion and Survival of Lactobacilli and Bifidobacteria on Table Olives with the Aim of Formulating a New Probiotic Food
Authors: Lavermicocca Paola , Valerio Francesca , Lonigro Stella Lisa , De Angelis Maria , Morelli Lorenzo , Callegari Maria Luisa , Rizzello Carlo G. , Visconti Angelo
Published: August 2005

With the aim of developing new functional foods, a traditional product, the table olive, was used as a vehicle for incorporating probiotic bacterial species. Survival on table olives of
Lactobacillus rhamnosus
(three strains),
Lactobacillus paracasei
(two strains),
Bifidobacterium bifidum
(one strain), and
Bifidobacterium longum
(one strain) at room temperature was investigated. The results obtained using a selected olive sample demonstrated that bifidobacteria and one strain of
L. rhamnosus
(
Lactobacillus
GG) showed a good survival rate, with a recovery of about 10
6
CFU g
−1
after 30 days. The
Lactobacillus
GG population remained unvaried until the end of the experiment, while a slight decline (to about 10
5
CFU g
−1
) was observed for bifidobacteria. High viability, with more than 10
7
CFU g
−1
, was observed throughout the 3-month experiment for
L. paracasei
IMPC2.1. This strain, selected for its potential probiotic characteristics and for its lengthy survival on olives, was used to validate table olives as a carrier for transporting bacterial cells into the human gastrointestinal tract.
L. paracasei
IMPC2.1 was recovered from fecal samples in four out of five volunteers fed 10 to 15 olives per day carrying about 10
9
to 10
10
viable cells for 10 days.
Role for HtrA in Stress Induction and Virulence Potential in
Listeria monocytogenes
Authors: Stack Helena M. , Sleator Roy D. , Bowers Megan , Hill Colin , Gahan Cormac G. M.
Published: August 2005

In silico analysis of the
Listeria monocytogenes
genome revealed
lmo0292
, a gene predicted to encode a HtrA-like serine protease. A stable insertion mutant was constructed, revealing a requirement for
htrA
in the listerial response to heat, acid, and penicillin stress. Transcriptional analysis revealed that
htrA
is not induced in response to heat shock but is induced in response to low pH and penicillin G stress. Furthermore,
htrA
expression was shown to be dependent upon the LisRK two-component sensor-kinase, a system known to respond to changes in integrity of the cell envelope. In addition, we demonstrated that a second in-frame start codon, upstream of that previously annotated for
L. monocytogenes htrA
, incorporating a putative signal sequence appears to influence virulence potential. Finally, a significant virulence defect was observed for the
htrA
mutant, indicating that this gene is required for full virulence in mice. Our findings suggest that
L. monocytogenes lmo0292
encodes an HtrA-like serine protease that is not part of the classical heat shock response but is involved in stress responses and virulence.
Elucidation of a Carotenoid Biosynthesis Gene Cluster Encoding a Novel Enzyme, 2,2′-β-Hydroxylase, from
Brevundimonas
sp. Strain SD212 and Combinatorial Biosynthesis of New or Rare Xanthophylls
Authors: Nishida Yasuhiro , Adachi Kyoko , Kasai Hiroaki , Shizuri Yoshikazu , Shindo Kazutoshi , Sawabe Akiyoshi , Komemushi Sadao , Miki Wataru , Misawa Norihiko
Published: August 2005

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium
Brevundimonas
sp. strain SD212 by using a common
crtI
sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes,
crtW
,
crtY
,
crtI
,
crtB
,
crtE
,
idi
, and
crtZ
. The individual ORFs were functionally analyzed by complementation studies using
Escherichia coli
that accumulated various carotenoid precursors due to the presence of other bacterial
crt
genes. In addition to functionally identifying the known
crt
genes, we found that one (ORF11, named
crtG
) coded for a novel enzyme, carotenoid 2,2′-β-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this
crtG
gene was introduced into
E. coli
accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2′-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.
Discrimination of Enterobacterial Repetitive Intergenic Consensus PCR Types of
Campylobacter coli
and
Campylobacter jejuni
by Fourier Transform Infrared Spectroscopy
Authors: Mouwen D. J. M. , Weijtens M. J. B. M. , Capita R. , Alonso-Calleja C. , Prieto M.
Published: August 2005

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species
Campylobacter coli
and
Campylobacter jejuni
and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species
Campylobacter coli
and
C. jejuni
, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm
−1
(described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic
Campylobacter
isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.
Responses of Baltic Sea Ice and Open-Water Natural Bacterial Communities to Salinity Change
Authors: Kaartokallio Hermanni , Laamanen Maria , Sivonen Kaarina
Published: August 2005

To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0°C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes with sequencing of DGGE bands from initial communities and communities of day 10 or 13 of the experiment. The sea ice-derived bacterial community was metabolically more active than the open-water community at the start of the experiment. Ice-derived bacterial communities were able to adapt to salinity change with smaller effects on physiology and community structure, whereas in the open-water bacterial communities, the bacterial cell volume evolution, bacterial abundance, and community structure responses indicated the presence of salinity stress. The closest relatives for all eight partial 16S rRNA gene sequences obtained were either organisms found in polar sea ice and other cold habitats or those found in summertime Baltic seawater. All sequences except one were associated with the α- and γ-proteobacteria or the
Cytophaga
-
Flavobacterium-Bacteroides
group. The overall physiological and community structure responses were parallel in ice-derived and open-water bacterial assemblages, which points to a linkage between community structure and physiology. These results support previous assumptions of the role of salinity fluctuation as a major selective factor shaping the sea ice bacterial community structure.
Genetic Characterization of Accumulation of Polyhydroxyalkanoate from Styrene in
Pseudomonas putida
CA-3
Authors: O'Leary Niall D. , O'Connor Kevin E. , Ward Patrick , Goff Miriam , Dobson Alan D. W.
Published: August 2005

Pseudomonas putida
CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn
5
random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in
P. putida
CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the
sty
operon in
P. putida
CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (
phaG
) gene in
P. putida
CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from
P. putida
KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with
P. putida
CA-3, maximal
phaG
expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, β-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in
P. putida
CA-3. Cloning and functional characterization of the
pha
locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.
Lactobacillus oligofermentans
sp. nov., Associated with Spoilage of Modified-Atmosphere-Packaged Poultry Products
Authors: Koort Joanna , Murros Anna , Coenye Tom , Eerola Susanna , Vandamme Peter , Sukura Antti , Björkroth Johanna
Published: August 2005

Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new
Lactobacillus
species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the
Lactobacillus durianis
type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the
Lactobacillus reuteri
group, which also includes
L. durianis
. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the
L. reuteri
group reference strains. The DNA-DNA hybridization levels between
Lactobacillus
sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the
L. durianis
type strain confirming that these isolates belong to the same species different from
L. durianis
. The name
Lactobacillus oligofermentans
sp. nov. is proposed, with strain LMG 22743
T
(also known as DSM 15707
T
or AMKR18
T
) as the type strain.
Distribution of
Cryptosporidium
Genotypes in Storm Event Water Samples from Three Watersheds in New York
Authors: Jiang Jianlin , Alderisio Kerri A. , Xiao Lihua
Published: August 2005

To assess the source and public health significance of
Cryptosporidium
oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of
Cryptosporidium
in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition,
C. hominis
(W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22
Cryptosporidium
species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several
Cryptosporidium
spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for
Cryptosporidium
by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of
Cryptosporidium
oocysts found in water.
Host Species-Specific Metabolic Fingerprint Database for Enterococci and
Escherichia coli
and Its Application To Identify Sources of Fecal Contamination in Surface Waters
Authors: Ahmed W. , Neller R. , Katouli M.
Published: August 2005

A metabolic fingerprint database of enterococci and
Escherichia coli
from 10 host groups of animals was developed to trace the sources of fecal contamination in surface waters. In all, 526 biochemical phenotypes (BPTs) of enterococci and 530
E. coli
BPTs were obtained from 4,057 enterococci and 3,728
E. coli
isolates tested. Of these, 231
Enterococcus
BPTs and 257
E. coli
BPTs were found in multiple host groups. The remaining 295
Enterococcus
BPTs and 273
E. coli
BPTs were unique to individual host groups. The database was used to trace the sources of fecal contamination in a local creek. The mean diversities (Di) of enterococci (Di = 0.76 ± 0.05) and
E. coli
(Di = 0.88 ± 0.04) were high (maximum 1) in water samples, indicating diverse sources of fecal contamination. Overall, 71% of BPTs of enterococci and 67% of
E. coli
BPTs from water samples were identified as human and animal sources. Altogether, 248
Enterococcus
BPTs and 282
E. coli
BPTs were found in water samples. Among enterococci, 26 (10%) BPTs were identical to those of humans and 152 BPTs (61%) were identical to those of animals (animal BPTs). Among
E. coli
isolates, 36 (13%) BPTs were identical to those of humans and 151 (54%) BPTs were identical to those of animals. Of the animal BPTs, 101 (66%)
Enterococcus
BPTs and 93 (62%)
E. coli
BPTs were also unique to individual animal groups. On the basis of these unique
Enterococcus
BPTs, chickens contributed 14% of contamination, followed by humans (10%), dogs (7%), and horses (6%). For
E. coli
, humans contributed 13% of contamination, followed by ducks (9%), cattle (7%), and chickens (6%). The developed metabolic fingerprint database was able to distinguish between human and animal sources as well as among animal species in the studied catchment.
Identification and Characterization of Putative Virulence Genes and Gene Clusters in
Aeromonas hydrophila
PPD134/91
Authors: Yu H. B. , Zhang Y. L. , Lau Y. L. , Yao F. , Vilches S. , Merino S. , Tomas J. M. , Howard S. P. , Leung K. Y.
Published: August 2005

Aeromonas hydrophila
is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of
A. hydrophila
is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in
A. hydrophila
PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of
A. hydrophila
PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in
A. hydrophila
pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in
A. hydrophila
. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.
Isolation and Characterization of a Novel Single-Stranded RNA Virus Infectious to a Marine Fungoid Protist,
Schizochytrium
sp. (Thraustochytriaceae, Labyrinthulea)
Authors: Takao Yoshitake , Nagasaki Keizo , Mise Kazuyuki , Okuno Tetsuro , Honda Daiske
Published: August 2005

Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist
Schizochytrium
sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 × 10
3
to 6.4 × 10
4
infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family
Dicistroviridae
based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms.
Low Intraspecific Diversity in a
Polynucleobacter
Subcluster Population Numerically Dominating Bacterioplankton of a Freshwater Pond
Authors: Hahn Martin W. , Pöckl Matthias , Wu Qinglong L.
Published: August 2005

Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a
Polynucleobacter
subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the
Polynucleobacter
cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a
Polynucleobacter
population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.
Structure and Topology of Microbial Communities in the Major Gut Compartments ofMelolontha melolonthaLarvae (Coleoptera: Scarabaeidae)
Authors: Egert Markus , Stingl Ulrich , Dyhrberg Bruun Lars , Pommerenke Bianca , Brune Andreas , Friedrich Michael W.
Published: August 2005

Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O2, H2, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut.Methanobrevibacterspp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to theClostridiales. Clones related to theActinobacteria,Bacillales,Lactobacillales, and γ-Proteobacteriawere restricted to the lumen, whereas clones related to the β- and δ-Proteobacteriawere found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented thatDesulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5′-phosphosulfate reductase geneapsAto DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.
Mycosubtilin Overproduction by
Bacillus subtilis
BBG100 Enhances the Organism's Antagonistic and Biocontrol Activities
Authors: Leclère Valérie , Béchet Max , Adam Akram , Guez Jean-Sébastien , Wathelet Bernard , Ongena Marc , Thonart Philippe , Gancel Frédérique , Chollet-Imbert Marlène , Jacques Philippe
Published: August 2005

A
Bacillus subtilis
derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene
repU
of the
Staphylococcus aureus
plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a
Pythium
infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.
16S rRNA Sequences and Differences in Bacteria Isolated from the Muztag Ata Glacier at Increasing Depths
Authors: Xiang Shurong , Yao Tandong , An Lizhe , Xu Bingliang , Wang Junxia
Published: August 2005

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates
recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs
Plateau (China). Only 11% were psychrophiles (grew at 2°C or −2°C up to ∼20°C), although the
majority (82%) were psychrotolerant (grew at 2°C or −2°C up to 37°C). The majority of the isolates
had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on
their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% wereγ
-
Proteobacteria
, 14.7% were α-
Proteobacteria
, 14.7% were
Flavobacteria
,
and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with
Proteobacteria
, HGC/
Cytophaga
-
Flavobacterium
-
Bacteroides
(CFB),
Proteobacteria
, LGC/CFB/HGC,
Cryobacterium psychrophilum
, HGC/CFB,
Proteobacteria
/HGC/CFB, and
HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below
the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting
the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and
nutrient availability within the ice.
Diurnal Variation of Cell Proliferation in Three Bacterial Taxa from Coastal North Sea Waters
Authors: Pernthaler Annelie , Pernthaler Jakob
Published: August 2005

Pulse-labeling with bromodeoxyuridine (BrdU) in combination with fluorescence in situ hybridization was applied to quantify the percentage of proliferating cells in coastal North Sea waters. In order to assess diurnal variability, we sampled eight or nine times, respectively, within 3 consecutive days at two seasons. Bacteria affiliated with the
Roseobacter
, SAR86, and NOR5 lineages constituted on average 19% ± 3%, 8% ± 2%, and 6% ± 1% of all cells in May 2002 and 17% ± 3%, 10% ± 2%, and 11% ± 3% in August. The relative abundances of the three populations either remained stable, or they changed very gradually during the observation periods. On average, 38 and 39% of all
Bacteria
exhibited DNA de novo synthesis in May and August, respectively. The fractions of proliferating cells in bacteria of the SAR86 (May, 59%; August, 72%) and the
Roseobacter
(48 and 53%) lineages were significantly above the community average. A substantial cell proliferation of population NOR5 (34%) was only encountered in August, concomitant with a dinoflagellate bloom. Significant short-term fluctuations of DNA-synthesizing cells were observed in
Roseobacter
during May and in NOR5 during August, hinting at a pronounced (temporal or spatial) mesoscale patchiness of growth rates in these populations. Since the BrdU proliferation assay is susceptible to misinterpretation, we also modeled the expected number of labeled cells at increasing BrdU incubation times in a slowly growing bacterial population. We suggest that the absence of visible DNA synthesis in marine bacterioplankton cells after DNA pulse-labeling must not be interpreted as an indication of cell “inactivity.”
Statistical Analyses: Possible Reasons for Unreliability of Source Tracking Efforts
Authors: Lasalde Clarivel , Rodríguez Roberto , Toranzos Gary A.
Published: August 2005

Analyses for the presence of indicator organisms provide information on the microbiological quality of water. Indicator organisms recommended by the United States Environmental Protection Agency for monitoring the microbiological quality of water include
Escherichia coli
, a thermotolerant coliform found in the feces of warm-blooded animals. These bacteria can also be isolated from environmental sources such as the recreational and pristine waters of tropical rain forests in the absence of fecal contamination. In the present study,
E. coli
isolates were compared to
E. coli
K12 (ATCC 29425) by restriction fragment length polymorphism using pulsed-field gel electrophoresis. Theoretically, genomic DNA patterns generated by PFGE are highly specific for the different isolates of an organism and can be used to identify variability between environmental and fecal isolates. Our results indicate a different band pattern for almost every one of the
E. coli
isolates analyzed. Cluster analysis did not show any relations between isolates and their source of origin. Only the discriminant function analysis grouped the samples with the source of origin. The discrepancy observed between the cluster analysis and discriminant function analysis relies on their mathematical basis. Our validation analyses indicate the presence of an artifact (i.e., grouping of environmental versus fecal samples as a product of the statistical analyses used and not as a result of separation in terms of source of origin) in the classification results; therefore, the large genetic heterogeneity observed in these
E. coli
populations makes the grouping of isolates by source rather difficult, if not impossible.
Regiospecificity of Two Multicomponent Monooxygenases from
Pseudomonas stutzeri
OX1: Molecular Basis for Catabolic Adaptation of This Microorganism to Methylated Aromatic Compounds
Authors: Cafaro Valeria , Notomista Eugenio , Capasso Paola , Di Donato Alberto
Published: August 2005

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with
Pseudomonas stutzeri
OX1 suggested that the
tou
operon coding for toluene
o
-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the
ph
operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and
o
-xylene oxidation using
Escherichia coli
cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and
o
-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is
p
-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on
o
-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by
P. stutzeri
OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and
o
-xylene.
Single-Nucleotide Polymorphism Phylotyping of
Escherichia coli
Authors: Hommais Florence , Pereira Sabrina , Acquaviva Cécile , Escobar-Páramo Patricia , Denamur Erick
Published: August 2005

We describe a rapid and easily automated phylogenetic grouping technique based on analysis of bacterial genome single-nucleotide polymorphisms (SNPs). We selected 13 SNPs derived from a complete sequence analysis of 11 essential genes previously used for multilocus sequence typing (MLST) of 30
Escherichia coli
strains representing the genetic diversity of the species. The 13 SNPs were localized in five genes,
trpA
,
trpB
,
putP
,
icdA
, and
polB
, and were selected to allow recovery of the main phylogenetic groups (groups A, B1, E, D, and B2) and subgroups of the species. In the first step, we validated the SNP approach in silico by extracting SNP data from the complete sequences of the five genes for a panel of 65 pathogenic strains belonging to different
E. coli
pathovars, which were previously analyzed by MLST. In the second step, we determined these SNPs by dideoxy single-base extension of unlabeled oligonucleotide primers for a collection of 183 commensal and extraintestinal clinical
E. coli
isolates and compared the SNP phylotyping method to previous well-established typing methods. This SNP phylotyping method proved to be consistent with the other methods for assigning phylogenetic groups to the different
E. coli
strains. In contrast to the other typing methods, such as multilocus enzyme electrophoresis, ribotyping, or PCR phylotyping using the presence/absence of three genomic DNA fragments, the SNP typing method described here is derived from a solid phylogenetic analysis, and the results obtained by this method are more meaningful. Our results indicate that similar approaches may be used for a wide variety of bacterial species.
Characterization of Colony Morphology Variants Isolated from
Pseudomonas aeruginosa
Biofilms
Authors: Kirisits Mary Jo , Prost Lynne , Starkey Melissa , Parsek Matthew R.
Published: August 2005

In this study, we report the isolation of small, rough, strongly cohesive colony morphology variants from aging
Pseudomonas aeruginosa
PAO1 biofilms. Similar to many of the
P. aeruginosa
colony morphology variants previously described in the literature, these variants autoaggregate in liquid culture and hyperadhere to solid surfaces. They also exhibit increased hydrophobicity and reduced motility compared to the wild-type parent strain. Despite the similarities in appearance of our colony morphology variant isolates on solid medium, the isolates showed a range of responses in various phenotypic assays. These variants form biofilms with significant three-dimensional structure and more biomass than the wild-type parent. To further explore the nature of the variants, their transcriptional profiles were evaluated. The variants generally showed increased expression of the
psl
and
pel
loci, which have been previously implicated in the adherence of
P. aeruginosa
to solid surfaces. When a mutation in the
psl
locus was introduced into a colony morphology variant, the colony morphology was only partially affected, but hyperadherence and autoaggregation were lost. Finally, similar colony morphology variants were found in isolates from cystic fibrosis patients. These variants displayed many of the same characteristics as the laboratory variants, suggesting a link between laboratory and cystic fibrosis biofilms.
Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge
Discodermia dissoluta
Authors: Schirmer Andreas , Gadkari Rishali , Reeves Christopher D. , Ibrahim Fadia , DeLong Edward F. , Hutchinson C. Richard
Published: August 2005

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge
Discodermia dissoluta
revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of
D. dissoluta
consists mainly of
Entotheonella
spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.
Adhesion of
Vibrio cholerae
to Granular Starches
Authors: Gancz Hanan , Niderman-Meyer Orly , Broza Meir , Kashi Yechezkel , Shimoni Eyal
Published: August 2005

Cholera is a severe diarrheal disease caused by specific serogroups of
Vibrio cholerae
that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that
V. cholerae
adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of
V. cholerae
and starch granules. Sugars metabolized by
V. cholerae
showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing
V. cholerae
adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.
Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of
Xylella fastidiosa
Authors: Lin Hong , Civerolo Edwin L. , Hu Rong , Barros Samuel , Francis Marta , Walker M. Andrew
Published: August 2005

A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of
Xylella fastidiosa
(strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the
X. fastidiosa
genome, clearly differentiated and clustered
X. fastidiosa
strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying
X. fastidiosa
epidemiology and population genetics.