bluuebunny/crossref_metadata_2025 huggingface dataset:
Dark Structures in Molecular Radiationless Transitions Determined by Ultrafast Diffraction
Authors: Srinivasan Ramesh , Feenstra Jonathan S. , Park Sang Tae , Xu Shoujun , Zewail Ahmed H.
Published: January 2005

The intermediate structures formed through radiationless transitions are termed “dark” because their existence is inferred indirectly from radiative transitions. We used ultrafast electron diffraction to directly determine these transient structures on both ground-state and excited-state potential energy surfaces of several aromatic molecules. The resolution in space and time (0.01 angstrom and 1 picosecond) enables differentiation between competing nonradiative pathways of bond breaking, vibronic coupling, and spin transition. For the systems reported here, the results reveal unexpected dynamical behavior. The observed ring opening of the structure depends on molecular substituents. This, together with the parallel bifurcation into physical and chemical channels, redefines structural dynamics of the energy landscape in radiationless processes.
From Stars to Dust: Looking into a Circumstellar Disk Through Chondritic Meteorites
Authors: Connolly Harold C.
Published: January 2005

One of the most fundamental questions in planetary science is, How did the solar system form? In this special issue, astronomical observations and theories constraining circumstellar disks, their lifetimes, and the formation of planetary to subplanetary objects are reviewed. At present, it is difficult to observe what is happening within disks and to determine if another disk environment is comparable to the early solar system disk environment (called the protoplanetary disk). Fortunately, we have chondritic meteorites, which provide a record of the processes that operated and materials present within the protoplanetary disk.
Detection of AirborneStachybotrys chartarumMacrocyclic Trichothecene Mycotoxins on Particulates Smaller than Conidia
Authors: Brasel T. L. , Douglas D. R. , Wilson S. C. , Straus D. C.
Published: January 2005

Highly respirable particles (diameter, <1 μm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborneStachybotrys chartarumtrichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluentStachybotrysgrowth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P< 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P< 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show thatS. chartarumtrichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.
Seasonal Change in Bacterial Flora and Biomass in Mountain Snow from the Tateyama Mountains, Japan, Analyzed by 16S rRNA Gene Sequencing and Real-Time PCR
Authors: Segawa Takahiro , Miyamoto Koji , Ushida Kazunari , Agata Kiyokazu , Okada Norihiro , Kohshima Shiro
Published: January 2005

The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium,
Cryobacterium psychrophilum
, and two psychrotrophic bacteria,
Variovorax paradoxus
and
Janthinobacterium lividum
. Bacterial colonies that developed in an in situ meltwater medium at 4°C were revealed to be
V. paradoxus
. The biomasses of
C. psychrophilum
,
J. lividum
, and
V. paradoxus
, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 × 10
5
-fold, 1.5 × 10
5
-fold, and 1.0 × 10
4
-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of
C. psychrophilum
and
J. lividum
increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of
V. paradoxus
did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.
Epidemiology ofCryptosporidium molnariin Spanish Gilthead Sea Bream (Sparus aurataL.) and European Sea Bass (Dicentrarchus labraxL.) Cultures: from Hatchery to Market Size
Authors: Sitjà-Bobadilla A. , Padrós F. , Aguilera C. , Alvarez-Pellitero P.
Published: January 2005

A long-term epidemiological study ofCryptosporidium molnariin aquacultured European sea bass (ESB) and gilthead sea bream (GSB) was performed in different types of facilities on the Atlantic, Cantabric, and Mediterranean coasts. Four types of studies were carried out. In study A, fish raised from juveniles to marketable size (ongrowing stage) were periodically sampled in three different types of cultures. Studies B and C focused on hatchery and nursery facilities. In study D, occasional samplings were performed during mortality or morbidity outbreaks. As a general trend,C. molnariwas more prevalent in GSB than in ESB. Data on the distribution pattern ofC. molnariin total sampled GSB (studies A, B, and D) had a variance higher than the mean (overdispersion). In GSB (study A), the type of ongrowing system (sea cages, earth ponds, or indoor tanks) was found to have no significant effect. There was a significant relationship between the presence of the parasite and both fish weight and season. The highest infection values were recorded in spring. Prevalence and intensity had convex weight profiles, with a peak in 30- to 100-g fish. In study D, the prevalence of infection was higher in fish recently introduced in sea cages and in preongrowing systems. In studies B and C, fish were almost never infected before entering the postlarval and nursery facilities. The parasite seems to enter the host mainly through the water in production steps with less stringent water treatment. Recirculation systems and fish cannibalism could contribute to oocyst concentration and dispersion in aquaculture facilities.
Seasonal Variation of
Ralstonia solanacearum
Biovar 2 Populations in a Spanish River: Recovery of Stressed Cells at Low Temperatures
Authors: Caruso Paola , Palomo Jose Luis , Bertolini Edson , Álvarez Belén , López María M. , Biosca Elena G.
Published: January 2005

The presence of
Ralstonia solanacearum
biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14°C or higher, but its isolation was usually unsuccessful at temperatures below 9°C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35°C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of
R. solanacearum
. Enrichment in MWB at both temperatures allowed us to recover
R. solanacearum
cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35°C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of
R. solanacearum
cells on solid media and their survival at low temperatures.
Prevalence and Antimicrobial Resistance of Thermophilic
Campylobacter
spp. from Cattle Farms in Washington State
Authors: Bae Wonki , Kaya Katherine N. , Hancock Dale D. , Call Douglas R. , Park Yong Ho , Besser Thomas E.
Published: January 2005

The prevalence of thermophilic
Campylobacter
spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic
Campylobacter
isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests,
hipO
colony blot hybridization, and multiplex
lpxA
PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution.
Campylobacter jejuni
was the most frequent species isolated (34.1%), followed by
Campylobacter coli
(7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types.
C. jejuni
was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates).
C. coli
isolates were more frequently resistant than
C. jejuni
, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in
C. coli
(51.5%) than in
C. jejuni
(5.1%). The results of this study demonstrate that
C. jejuni
is widely distributed among Washington cattle farms, while
C. coli
is more narrowly distributed but significantly more resistant.
Diversity of Green-Like and Red-Like Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes (
cbbL
) in Differently Managed Agricultural Soils
Authors: Selesi Draženka , Schmid Michael , Hartmann Anton
Published: January 2005

A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (
cbbL
) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of
cbbL
genes. The diversity of these
cbbL
genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment.
cbbL
gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique
cbbL
clones were sequenced and analyzed phylogenetically. The green-like
cbbL
sequences revealed a very low level of diversity, being closely related to the
cbbL
genes of
Nitrobacter winogradskyi
and
Nitrobacter vulgaris
. In contrast, the red-like
cbbL
gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like
cbbL
genes were only distantly related to already known
cbbL
sequences and even formed separate clusters. In order to extend the database of available red-like
cbbL
sequences, we amplified
cbbL
sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the
cbbL
gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as
Bacillus
,
Streptomyces
, and
Arthrobacter
harbor red-like
cbbL
genes which fall into the
cbbL
gene clusters retrieved from the investigated soils.
Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in
Corynebacterium glutamicum
Authors: Elišáková Veronika , Pátek Miroslav , Holátko Jiří , Nešvera Jan , Leyval Damien , Goergen Jean-Louis , Delaunay Stéphane
Published: January 2005

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole
Corynebacterium glutamicum ilvBNC
operon, coding for acetohydroxy acid synthase (
ilvBN
) and aceto hydroxy acid isomeroreductase (
ilvC
), was cloned in the newly constructed
Escherichia coli
-
C. glutamicum
shuttle vector pECKA (5.4 kb, Km
r
). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by
ilvN
. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the
ilvBNC
operon with mutations on the plasmid or the
ilvN
M13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the
ilvN
M13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free
C. glutamicum
Δ
ilvA
Δ
panB ilvN
M13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.
Geographic and Environmental Sources of Variation in Lake Bacterial Community Composition
Authors: Yannarell Anthony C. , Triplett Eric W.
Published: January 2005

This study used a genetic fingerprinting technique (automated ribosomal intergenic spacer analysis [ARISA]) to characterize microbial communities from a culture-independent perspective and to identify those environmental factors that influence the diversity of bacterial assemblages in Wisconsin lakes. The relationships between bacterial community composition and 11 environmental variables for a suite of 30 lakes from northern and southern Wisconsin were explored by canonical correspondence analysis (CCA). In addition, the study assessed the influences of ARISA fragment detection threshold (sensitivity) and the quantitative, semiquantitative, and binary (presence-absence) use of ARISA data. It was determined that the sensitivity of ARISA was influential only when presence-absence-transformed data were used. The outcomes of analyses depended somewhat on the data transformation applied to ARISA data, but there were some features common to all of the CCA models. These commonalities indicated that differences in bacterial communities were best explained by regional (i.e., northern versus southern Wisconsin lakes) and landscape level (i.e., seepage lakes versus drainage lakes) factors. ARISA profiles from May samples were consistently different from those collected in other months. In addition, communities varied along gradients of pH and water clarity (Secchi depth) both within and among regions. The results demonstrate that environmental, temporal, regional, and landscape level features interact to determine the makeup of bacterial assemblages in northern temperate lakes.
Quantification of Ammonia-Oxidizing Bacteria and Factors Controlling Nitrification in Salt Marsh Sediments
Authors: Dollhopf Sherry L. , Hyun Jung-Ho , Smith April C. , Adams Harold J. , O'Brien Sean , Kostka Joel E.
Published: January 2005

To elucidate the geomicrobiological factors controlling nitrification in salt marsh sediments, a comprehensive approach involving sediment geochemistry, process rate measurements, and quantification of the genetic potential for nitrification was applied to three contrasting salt marsh habitats: areas colonized by the tall (TS) or short (SS) form of
Spartina alterniflora
and unvegetated creek banks (CBs). Nitrification and denitrification potential rates were strongly correlated with one another and with macrofaunal burrow abundance, indicating that coupled nitrification-denitrification was enhanced by macrofaunal burrowing activity. Ammonia monooxygenase (
amoA
) gene copy numbers were used to estimate the ammonia-oxidizing bacterial population size (5.6 × 10
4
to 1.3 × 10
6
g of wet sediment
−1
), which correlated with nitrification potentials and was 1 order of magnitude higher for TS and CB than for SS. TS and CB sediments also had higher Fe(III) content, higher Fe(III)-to-total reduced sulfur ratios, higher Fe(III) reduction rates, and lower dissolved sulfides than SS sediments. Iron(III) content and reduction rates were positively correlated with nitrification and denitrification potential and
amoA
gene copy number. Laboratory slurry incubations supported field data, confirming that increased amounts of Fe(III) relieved sulfide inhibition of nitrification. We propose that macrofaunal burrowing and high concentrations of Fe(III) stimulate nitrifying bacterial populations, and thus may increase nitrogen removal through coupled nitrification-denitrification in salt marsh sediments.
A
clp
Gene Homologue Belonging to the Crp Gene Family Globally Regulates Lytic Enzyme Production, Antimicrobial Activity, and Biological Control Activity Expressed by
Lysobacter enzymogenes
Strain C3
Authors: Kobayashi Donald Y. , Reedy Ralph M. , Palumbo Jeffrey D. , Zhou Jun-Ma , Yuen Gary Y.
Published: January 2005

Lysobacter enzymogenes
strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and to evaluate their biocontrol efficacy. A single mini-Tn
5-lacZ
1
-cat
transposon mutant of
L. enzymogenes
strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a
clp
gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related
N
-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with
clp
. Chromosomal deletion mutants with mutations in
clp
and between
clp
and the acetyltransferase gene verified the 5E4 mutant phenotype. The
clp
gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain.
clp
mutant strains were significantly affected in biological control of
pythium
damping-off of sugar beet and
bipolaris
leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that
clp
is a global regulatory gene that controls biocontrol traits expressed by
L. enzymogenes
C3.
Different Inactivation Behaviors of MS-2 Phage and
Escherichia coli
in TiO
2
Photocatalytic Disinfection
Authors: Cho Min , Chung Hyenmi , Choi Wonyong , Yoon Jeyong
Published: January 2005

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO
2
photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO
2
, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H
2
O
2
and O
2
·
−
, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and
Escherichia coli
as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction,
para
-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers,
tert
-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe
2+
, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that
E. coli
is inactivated by both the free and the surface-bound hydroxyl radicals.
E. coli
might also be inactivated by other ROS, such as O
2
·
−
and H
2
O
2
, according to the present results.
Development and Validation of PCR Primers To Assess the Diversity of
Clostridium
spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis
Authors: Le Bourhis Anne-Gaëlle , Saunier Katiana , Doré Joël , Carlier Jean-Philippe , Chamba Jean-François , Popoff Michel-Robert , Tholozan Jean-Luc
Published: January 2005

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus
Clostridium
(the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of
Clostridium
spp. was confirmed by sequencing of excised bands.
C. tyrobutyricum
and
C. beijerinckii
contaminated 15 and 14 of the 20 cheese samples tested, respectively, and
C. butyricum
and
C. sporogenes
were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species,
C. tyrobutyricum
and
C. sporogenes
, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of
C. tyrobutyricum
DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify
Clostridium
in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.
Identification, Cloning, and Characterization of a
Lactococcus lactis
Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation
Authors: Smit Bart A. , van Hylckama Vlieg Johan E. T. , Engels Wim J. M. , Meijer Laura , Wouters Jan T. M. , Smit Gerrit
Published: January 2005

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of
Lactococcus lactis
strains. By using a random mutagenesis approach, the gene encoding KdcA in
L. lactis
B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated
ipd
, which encodes a putative indole pyruvate decarboxylase, in
L. lactis
IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the
ipd
gene encoding a truncated nonfunctional decarboxylase. The
kdcA
gene was overexpressed in
L. lactis
for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.
Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis
Authors: Hashimoto Shinji , Ogura Mayumi , Aritomi Kazuo , Hoshida Hisashi , Nishizawa Yoshinori , Akada Rinji
Published: January 2005

Auxotrophic mutants of the yeast
Saccharomyces cerevisiae
are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His
−
, Met
−
, Lys
−
, Trp
−
, Leu
−
, Arg
−
, and Ura
−
auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With
HIS3
as a selectable marker, the yeast
TDH3
overexpression promoter was inserted upstream of
ATF1
, encoding alcohol acetyltransferase, by one-step gene replacement in a
his3
mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.
Inactivation of
Escherichia coli
O157:H7 in Simulated Human Gastric Fluid
Authors: Tamplin Mark L.
Published: January 2005

Human disease caused by
Escherichia coli
O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of
E. coli
O157:H7 survival in gastric fluid, individual
E. coli
O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37°C for up to 4 h. The highest inactivation rate among nine
E. coli
O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were −1.344 ± 0.564 and −0.997 ± 0.388 log
10
CFU/h, respectively. In contrast, the average inactivation rate for 10
E. coli
O157:H7 strains suspended in 10:1 SGF-CGB was −0.081 ± 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for
Shigella flexneri
strain 5348 in 100:1 and 10:1 SGF-saline was −8.784 and −17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for
E. coli
O157:H7 strains in SGF-saline and were 4-fold higher than those for
E. coli
O157:H7 strains in SGF-CGB. The survival rate of
E. coli
O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting
E. coli
O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for
E. coli
O157:H7 and to evaluate potential surrogate organisms.
High-Pressure Inactivation of Hepatitis A Virus within Oysters
Authors: Calci Kevin R. , Meade Gloria K. , Tezloff Robert C. , Kingsley David H.
Published: January 2005

Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log
10
were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3°C. Bioconcentration of nearly 6 log
10
PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.
Interactions between Oral Bacteria: Inhibition of
Streptococcus mutans
Bacteriocin Production by
Streptococcus gordonii
Authors: Wang Bing-Yan , Kuramitsu Howard K.
Published: January 2005

Streptococcus mutans
has been recognized as an important etiological agent in human dental caries. Some strains of
S. mutans
also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by
S. mutans
strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the
com
genes. We also demonstrated that interactions with some other oral streptococci interfered with
S. mutans
bacteriocin production both in broth and in biofilms. The inhibition of
S. mutans
bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn
916
mutagenesis, we identified a gene (
sgc
; named for
Streptococcus gordonii
challisin) responsible for the inhibition of
S. mutans
bacteriocin production by
S. gordonii
Challis. Interruption of the
sgc
gene in
S. gordonii
Challis resulted in attenuated inhibition of
S. mutans
bacteriocin production. The supernatant fluids from the
sgc
mutant did not inactivate the exogenous
S. mutans
CSP as did those from the parent strain Challis.
S. gordonii
Challis did not inactivate bacteriocin produced by
S. mutans
GS5. Because
S. mutans
uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.
Bacillus subtilis
α-Phosphoglucomutase Is Required for Normal Cell Morphology and Biofilm Formation
Authors: Lazarevic Vladimir , Soldo Blazenka , Médico Noël , Pooley Harold , Bron Sierd , Karamata Dimitri
Published: January 2005

Mutations designated
gtaC
and
gtaE
that affect α-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and α-glucose 1-phosphate were mapped to the
Bacillus subtilis pgcA
(
yhxB
) gene. Backcrossing of the two mutations into the 168 reference strain was accompanied by impaired α-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages φ29 and ρ11. Altered cell morphology, reversible by additional magnesium ions, may be correlated with a deficiency in the membrane glycolipid. The deficiency in biofilm formation in
gtaC
and
gtaE
mutants may be attributed to an inability to synthesize UDP-glucose, an important intermediate in a number of cell envelope biosynthetic processes.
Use of Gradient Plates To Study Combined Effects of Temperature, pH, and NaCl Concentration on Growth of
Monascus ruber
van Tieghem, an Ascomycetes Fungus Isolated from Green Table Olives
Authors: Panagou E. Z. , Skandamis P. N. , Nychas G.-J. E.
Published: January 2005

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus
Monascus ruber
van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35°C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for
P
values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35°C, whereas at 35°C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.
Effect of Direct Electric Current on the Cell Surface Properties of Phenol-Degrading Bacteria
Authors: Luo Qishi , Wang Hui , Zhang Xihui , Qian Yi
Published: January 2005

The change in cell surface properties in the presence of electric currents is of critical concern when the potential to manipulate bacterial movement with electric fields is evaluated. In this study, the effects of different direct electric currents on the cell surface properties involved in bacterial adhesion were investigated by using a mixed phenol-degrading bacterial culture in the exponential growth phase. The traits investigated were surface hydrophobicity (measured by adherence to
n
-octane), net surface electrostatic charge (determined by measurement of the zeta potential), and the cell surface shape and polymers (determined by scanning electron microscope analysis). The results showed that a lower current (less than 20 mA) induced no significant changes in the surface properties of phenol-degrading bacteria, that an electric current of 20 mA could increase the surface hydrophobicity and flatten the cell shape, and that a higher current (40 mA) could increase the surface extracellular substances and the net negative surface electrostatic charge. The results also revealed that the electric current effects on cell hydrophobicity varied with the suspending medium. We suggest that an electric current greater than 20 mA is not suitable for use in manipulation of the movement of the phenol-degrading bacteria, although such a current might favor the electrophoretic movement of the bacterial species.
Use of Fluorophore-Conjugated Lectins To Study Cell-Cell Interactions in Model Marine Biofilms
Authors: Wigglesworth-Cooksey Barbara , Cooksey Keith E.
Published: January 2005

Biofilms dominated by pennate diatoms are important in fields as diverse as ship biofouling and marine littoral sediment stabilization. The architecture of a biofilm depends on the fact that much of its mass consists of extracellular polymers. Although most illuminated biofilms in nature are dominated by phototrophs, they also contain heterotrophic bacteria. Given the close spatial association of the two types of organisms, cell-cell interaction is likely. Fluorophore-conjugated lectins were used to demonstrate the sites of the various extracellular polymers in three species of diatoms. Based on their lectin staining properties, the polymers in different species appeared to be similar, but their involvement in the process of attachment to a surface differed. In a coculture
Pseudoalteromonas
sp. strain 4 or its sterilized spent medium reduced the ability of
Amphora coffeaeformis
and
Navicula
sp. strains 1 and D to adhere, inhibited motility, and caused agglutination and eventually diatom cell lysis. Diatoms could be protected from the negative effects of the bacterial spent medium if
d
-galactose or mannan was included in the incubation medium. The active principle of the spent medium is probably a lectin/agglutinin that is able to bind to the extracellular polymers of the diatoms that are involved in adhesion and motility. Awareness of interactions of this type is important in the study of natural biofilms.
Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo
Authors: Dolk Edward , van der Vaart Marcel , Lutje Hulsik David , Vriend Gert , de Haard Hans , Spinelli Silvia , Cambillau Christian , Frenken Leon , Verrips Theo
Published: January 2005

As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of
Malassezia furfur
in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of
M. furfur
, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications.
Simultaneous Recovery of Extracellular and Intracellular DNA Suitable for Molecular Studies from Marine Sediments
Authors: Corinaldesi Cinzia , Danovaro Roberto , Dell'Anno Antonio
Published: January 2005

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.
Diversity and Distribution of Methanotrophic Archaea at Cold Seeps
Authors: Knittel Katrin , Lösekann Tina , Boetius Antje , Kort Renate , Amann Rudolf
Published: January 2005

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to
Methanococcoides
spp.) and to the
Crenarchaeota
of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the
Desulfosarcina-Desulfococcus
branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at
Beggiatoa
sites (80% ANME-2a, 20% ANME-2c) and
Calyptogena
sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.
Nearly Identical Bacteriophage Structural Gene Sequences Are Widely Distributed in both Marine and Freshwater Environments
Authors: Short Cindy M. , Suttle Curtis A.
Published: January 2005

Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria.
Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism
Authors: Jernberg Cecilia , Sullivan Åsa , Edlund Charlotta , Jansson Janet K.
Published: January 2005

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as
Eubacterium
spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic
Lactobacillus
strains were easily tracked by T-RFLP and were shown to be the dominant
Lactobacillus
community members in the intestinal microflora of subjects who received the probiotic.