bluuebunny/crossref_metadata_2025 huggingface dataset:
Enteroviruses and Bacteriophages in Bathing Waters
Authors: Mocé-Llivina Laura , Lucena Francisco , Jofre Juan
Published: November 2005

A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages were also tested. Cultivable enteroviruses were detected in 55% of the samples, most of which complied with bacteriological criteria. In contrast, viral genomes were only detected in 20% of the samples by reverse transcription-PCR. Somatic coliphages outnumbered all other indicators. F-specific RNA phages were detected in only 15% of the samples, whereas phages infecting
Bacteroides thetaiotaomicron
were detected in 70% of samples. A numerical relationship between the numbers of enteroviruses and the numbers of enterococci and somatic coliphages was observed. In situ inactivation experiments showed that viruses persisted significantly longer than the bacterial indicators. Only somatic coliphages and bacteriophages infecting
Bacteroides
persisted longer than the viruses. These results explain the numbers of enteroviruses and indicators in bathing waters attending the numbers usually found in sewage in the area. Somatic coliphages show a very good potential to predict the risk of viruses being present in bathing waters.
Identification and Characterization of a Novel Biotin Biosynthesis Gene inSaccharomyces cerevisiae
Authors: Wu Hong , Ito Kiyoshi , Shimoi Hitoshi
Published: November 2005

YeastSaccharomyces cerevisiaecells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified asS. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was namedBIO6because it has 52% identity withBIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without theBIO6gene are auxotrophic for biotin, whereas yeasts holding theBIO6gene are prototrophic for biotin. TheBIO6gene was disrupted in strain A364A, which is a laboratory strain with one copy of theBIO6gene. Although strain A364A is prototrophic for biotin, aBIO6disrupted mutant was found to be auxotrophic for biotin. TheBIO6disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while thebio3disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of theBIO6gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that theBIO6gene is a novel biotin biosynthesis gene ofS. cerevisiae.
Development of a Rapid Method for Simultaneous Recovery of Diverse Microbes in Drinking Water by Ultrafiltration with Sodium Polyphosphate and Surfactants
Authors: Hill Vincent R. , Polaczyk Amy L. , Hahn Donghyun , Narayanan Jothikumar , Cromeans Theresa L. , Roberts Jacquelin M. , Amburgey James E.
Published: November 2005

The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2,
Escherichia coli
,
Enterococcus faecalis
,
Cryptosporidium parvum
, 4.5-μm microspheres,
Salmonella enterica
serovar Typhimurium,
Bacillus globigii
endospores, and echovirus 1. Ten-liter tap water samples were concentrated to ∼250 ml in 12 to 42 min, depending on the experimental condition. Initial experiments indicated that pretreating filters with fetal bovine serum or NaPP resulted in an increase in microbe recovery. The addition of NaPP to the tap water samples resulted in significantly higher microbe and microsphere recovery efficiencies. Backflushing of the ultrafilter was found to significantly improve recovery efficiencies. The effectiveness of backflushing was improved further with the addition of Tween 80 to the backflush solution. The ultrafiltration method developed in this study, incorporating the use of NaPP pretreatment and surfactant solution backflushing, was found to recover MS2,
C. parvum
, microspheres, and several bacterial species with mean recovery efficiencies of 70 to 93%. The mean recovery efficiency for echovirus 1 (49%) was the lowest of the microbes studied for this method. This research demonstrates that ultrafiltration can be effective for recovering diverse microbes simultaneously in tap water and that chemical dispersants and surfactants can be beneficial for improving microbial recovery using this technique.
Bacterial Populations Active in Metabolism of C
1
Compounds in the Sediment of Lake Washington, a Freshwater Lake
Authors: Nercessian Olivier , Noyes Emma , Kalyuzhnaya Marina G. , Lidstrom Mary E. , Chistoserdova Ludmila
Published: November 2005

Active
members of the bacterial community in the sediment of Lake Washington,
with special emphasis on C
1
utilizers, were identified by
employing two complementary culture-independent approaches: reverse
transcription of environmental mRNA and 16S rRNA combined with PCR
(RT-PCR) and stable-isotope probing (SIP) of DNA with the
13
C-labeled C
1
substrates methanol, methylamine,
formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of
16S rRNA-encoding genes revealed that gammaproteobacterial
methanotrophs belonging to
Methylobacter
and
Methylomonas
dominate the active methylotroph population,
while only one other known methylotrophic lineage,
Methylophilaceae
, was detected via this approach. Analysis of
RT-PCR-amplified functional genes,
pmoA
and
fae
,
allowed detection of alphaproteobacterial (
Methylosinus
) and
gammaproteobacterial (
Methylobacter
,
Methylomonas
,
and
Methylomicrobium
) methanotrophs, methylotrophs of
the genus
Methylobacterium
, and yet-unidentified
proteobacteria. SIP experiments allowed detection of a broad variety of
groups actively metabolizing C
1
compounds. Comparisons
between 16S rRNA gene pools amplified from
[
13
C]DNA and from [
12
C]DNA revealed that the
proportion of
Methylophilus
-related sequences increased in the
presence of [
13
C]methanol, [
13
C]methylamine, and
[
13
C]formaldehyde;
Novosphingobium
-related
sequences were enriched in the presence of [
13
C]methanol;
Gemmatimonadaceae
-related sequences were enriched in the
presence of [
13
C]formaldehyde and [
13
C]formate;
and
Xanthomonadaceae
-related sequences were enriched in the
presence of [
13
C]formate. Analysis of
fae
genes
amplified from [
13
C]DNAs isolated from different microcosms
revealed specific shifts in populations in response to a specific
C
1
compound:
Methylosinus
sequences dominated the
[
13
C]methanol microcosm pool, and beta- and
gammaproteobacterial sequences dominated the
[
13
C]methylamine microcosm pool. The
[
13
C]formaldehyde microcosm was dominated by
betaproteobacterial sequences and by sequences of a nonaffiliated
group, while the [
13
C]formate microcosm was dominated by
alpha- and betaproteobacterial sequences. Overall, these data point
toward the presence of a diverse population of active methylotrophs in
Lake Washington sediments and toward the existence of yet-uncultivated
organisms.
Efficient Procedure for Purification of ObligateIntracellular
Wolbachia pipientis
and RepresentativeAmplification of Its Genome by Multiple-DisplacementAmplification
Authors: Mavingui Patrick , Tran Van Van , Labeyrie Estelle , Rancès Edwige , Vavre Fabrice , Simonet Pascal
Published: November 2005

Bacteria
belonging to the genus
Wolbachia
are obligatorymicroendocytobionts that infect a variety of arthropods and a majority
of filarial nematode species, where they induce reproductive
alterations or establish a mutualistic symbiosis. Although two whole
genome sequences of
Wolbachia pipientis
, for strain wMel from
Drosophila melanogaster
and strain wBm from
Brugia
malayi
, have been fully completed and six other genome sequencing
projects are ongoing
(
http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin
),
genetic analyses of these bacteria are still scarce, mainly due to the
inability to cultivate them outside of eukaryotic cells. Usually, a
large amount of host tissue (a thousand individuals, or about
10 g) is required in order to purify
Wolbachia
and
extract its DNA, which is often recovered in small amounts and
contaminated by host cell DNA, thus hindering genomic studies. In this
report, we describe an efficient and reliable procedure to
representatively amplify the
Wolbachia
genome by
multiple-displacement amplification from limited infected host tissue
(0.2 g or 2 × 10
7
cells). We obtained sufficient
amounts (8 to 10 μg) of DNA of suitable quality for genomic
studies, and we demonstrated that the amplified DNA contained all of
the
Wolbachia
loci targeted. In addition, our data indicated
that the genome of strain wRi, an obligatory endosymbiont of
Drosophila simulans
, shares a similar overall architecture
with its relative strain
wMel.
Redox Fluctuation Structures Microbial Communities in a Wet Tropical Soil
Authors: Pett-Ridge J. , Firestone M. K.
Published: November 2005

Frequent high-amplitude redox fluctuation may be a strong selective force on the phylogenetic and physiological composition of soil bacterial communities and may promote metabolic plasticity or redox tolerance mechanisms. To determine effects of fluctuating oxygen regimens, we incubated tropical soils under four treatments: aerobic, anaerobic, 12-h oxic/anoxic fluctuation, and 4-day oxic/anoxic fluctuation. Changes in soil bacterial community structure and diversity were monitored with terminal restriction fragment length polymorphism (T-RFLP) fingerprints. These profiles were correlated with gross N cycling rates, and a Web-based phylogenetic assignment tool was used to infer putative community composition from multiple fragment patterns. T-RFLP ordinations indicated that bacterial communities from 4-day oxic/anoxic incubations were most similar to field communities, whereas those incubated under consistently aerobic or anaerobic regimens developed distinctly different molecular profiles. Terminal fragments found in field soils persisted either in 4-day fluctuation/aerobic conditions or in anaerobic/12-h treatments but rarely in both. Only 3 of 179 total fragments were ubiquitous in all soils. Soil bacterial communities inferred from in silico phylogenetic assignment appeared to be dominated byActinobacteria(especiallyMicrococcusandStreptomycetes), “Bacilli,” “Clostridia,” andBurkholderiaand lost significant diversity under consistently or frequently anoxic incubations. Community patterns correlated well with redox-sensitive processes such as nitrification, dissimilatory nitrate reduction to ammonium (DNRA), and denitrification but did not predict patterns of more general functions such as N mineralization and consumption. The results suggest that this soil's indigenous bacteria are highly adapted to fluctuating redox regimens and generally possess physiological tolerance mechanisms which allow them to withstand unfavorable redox periods.
Inactivation of
Bacillus
Endospores in Envelopes by Electron Beam Irradiation
Authors: Helfinstine Shannon L. , Vargas-Aburto Carlos , Uribe Roberto M. , Woolverton Christopher J.
Published: November 2005

The anthrax incidents in the United States in the fall of 2001 led to the use of electron beam (EB) processing to sanitize the mail for the U.S. Postal Service. This method of sanitization has prompted the need to further investigate the effect of EB irradiation on the destruction of
Bacillus
endospores. In this study, endospores of an anthrax surrogate,
B. atrophaeus
, were destroyed to demonstrate the efficacy of EB treatment of such biohazard spores. EB exposures were performed to determine (i) the inactivation of varying
B. atrophaeus
spore concentrations, (ii) a D
10
value (dose required to reduce a population by 1 log
10
) for the
B. atrophaeus
spores, (iii) the effects of spore survival at the bottom of a standardized paper envelope stack, and (iv) the maximum temperature received by spores. A maximum temperature of 49.2°C was reached at a lethal dose of ∼40 kGy, which is a significantly lower temperature than that needed to kill spores by thermal effects alone. A D
10
value of 1.53 kGy was determined for the species. A surface EB dose between 25 and 32 kGy produced the appropriate killing dose of EB between 11 and 16 kGy required to inactivate 8 log
10
spores, when spore samples were placed at the bottom of a 5.5-cm stack of envelopes.
European Origin of
Bradyrhizobium
Populations Infecting Lupins and Serradella in Soils of Western Australia and South Africa
Authors: Stępkowski Tomasz , Moulin Lionel , Krzyżańska Agnieszka , McInnes Alison , Law Ian J. , Howieson John
Published: November 2005

We applied a multilocus phylogenetic approach to elucidate the origin of serradella and lupin
Bradyrhizobium
strains that persist in soils of Western Australia and South Africa. The selected strains belonged to different randomly amplified polymorphic DNA (RAPD)-PCR clusters that were distinct from RAPD clusters of applied inoculant strains. Phylogenetic analyses were performed with nodulation genes (
nodA
,
nodZ
,
nolL
,
noeI
), housekeeping genes (
dnaK
,
recA
,
glnII
,
atpD
), and 16S-23S rRNA intergenic transcribed spacer sequences. Housekeeping gene phylogenies revealed that all serradella and
Lupinus cosentinii
isolates from Western Australia and three of five South African narrow-leaf lupin strains were intermingled with the strains of
Bradyrhizobium canariense
, forming a well supported branch on each of the trees. All
nodA
gene sequences of the lupin and serradella bradyrhizobia formed a single branch, referred to as clade II, together with the sequences of other lupin and serradella strains. Similar patterns were detected in
nodZ
and
nolL
trees. In contrast,
nodA
sequences of the strains isolated from native Australian legumes formed either a new branch called clade IV or belonged to clade I or III, whereas their nonsymbiotic genes grouped outside the
B. canariense
branch. These data suggest that the lupin and serradella strains, including the strains from uncultivated
L. cosentinii
plants, are descendants of strains that most likely were brought from Europe accidentally with lupin and serradella seeds. The observed dominance of
B. canariense
strains may be related to this species' adaptation to acid soils common in Western Australia and South Africa and, presumably, to their intrinsic ability to compete for nodulation of lupins and serradella.
Occurrence of
Mycobacterium avium
subsp.
paratuberculosis
in Untreated Water in Northern Ireland
Authors: Whan Lynne , Ball Hywel J. , Grant Irene R. , Rowe Michael T.
Published: November 2005

Mycobacterium avium
subsp.
paratuberculosis
is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for
M. avium
subsp.
paratuberculosis
in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested
M. avium
subsp.
paratuberculosis
positive by one or more of the three detection methods.
M. avium
subsp.
paratuberculosis
was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of
M. avium
subsp.
paratuberculosis
was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of
M. avium
subsp.
paratuberculosis
. In general, a higher incidence of
M. avium
subsp.
paratuberculosis
was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove
M. avium
subsp.
paratuberculosis
from untreated water and the possible risks posed by contact with recreational water sources.
Transcriptional Expression of the
tceA
Gene in a
Dehalococcoides-
Containing Microbial Enrichment
Authors: Johnson David R. , Lee Patrick K. H. , Holmes Victor F. , Fortin Alexander C. , Alvarez-Cohen Lisa
Published: November 2005

Dynamic changes in the transcriptional expression of the
tceA
gene, which encodes a trichloroethene reductive dehalogenase, were characterized in a
Dehalococcoides
-containing microbial enrichment culture. Expression was quantified by real-time PCR as the number of
tceA
transcripts per
tceA
gene. Expression of
tceA
increased 40-fold after chlorinated ethene-starved cells were exposed to trichloroethene (TCE),
cis
-dichloroethene (DCE), or 1,1-DCE but did not increase after exposure to tetrachloroethene or vinyl chloride. Surprisingly,
tceA
expression also increased 30-fold after cellular exposure to the nonmetabolic substrate
trans
-DCE, indicating that expression of
tceA
is induced by both growth-supporting and non-growth-supporting chlorinated ethenes. Additional experiments revealed that the level of
tceA
expression was independent of the concentration of chlorinated ethenes (sum concentrations of TCE and DCEs of 2.2 to 333 μM), the concentration of the electron donor hydrogen (concentrations of 12 nM to 17 μM), and the presence of alternate bacterial electron acceptors (5 mM concentrations of fumarate, sulfate, sulfite, thiosulfate, nitrate, or nitrite) but was highly dependent on incubation temperature.
Fate and Role of Ammonium Ions during Fermentation of Citric Acid by
Aspergillus niger
Authors: Papagianni Maria , Wayman Frank , Mattey Michael
Published: November 2005

Stoichiometric modeling of the early stages of the citric acid fermentation process by
Aspergillus niger
revealed that ammonium ions combine with a carbon-containing metabolite inside the cell, in a ratio 1:1, to form a nitrogen compound which is then excreted by the mycelium. High-performance liquid chromatography analysis identified glucosamine as the product of the relationship between glucose and ammonium during the early stages of the citric acid fermentation process. Slightly acidic internal pHs, extremely low ammonium ion concentrations inside the cell, and glucosamine synthesis come into direct contradiction with the earlier theory of the ammonium pool inside the cell, regarded as responsible for inhibition of the enzyme phosphofructokinase. At later fermentation stages, when the mycelium is involved in a process of fragmentation and regrowth, the addition of ammonium sulfate leads to a series of events: the formation and secretion of glucosamine in elevated amounts, the short inhibition of citrate synthesis, growth enhancement, the utilization of glucosamine, and finally, the enhancement of citric acid production rates. Obviously, the enzymatic processes underlining the phenomena need to be reexamined. As a by-product of the citric acid fermentation, glucosamine is reported for the first time here. Suitable process manipulations of the system described in this work could lead to successful glucosamine recovery at the point of its highest yield before degradation by the fungus occurs.
Prevalence ofBorrelia burgdorferiSensu Lato Genospecies inIxodes ricinusTicks in Europe: a Metaanalysis
Authors: Rauter Carolin , Hartung Thomas
Published: November 2005

In Europe,Borrelia burgdorferigenospecies causing Lyme borreliosis are mainly transmitted by the tickIxodes ricinus. Since its discovery,B. burgdorferihas been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies ofI. ricinusticks infected withB. burgdorferisensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection ofI. ricinuswithBorreliain Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection ofI. ricinuswithBorreliagenospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection ofI. ricinuswere found in countries in central Europe.B. afzeliiandB. gariniiare the most commonBorreliaspecies, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection byBorreliaspecies was found forB. gariniiandB. valaisiana.
Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) ofEscherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants
Authors: Lütke-Eversloh Tina , Stephanopoulos Gregory
Published: November 2005

In order to get insights into the feedback regulation by tyrosine of theEscherichia colichorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by thetyrAgene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. ThetyrAfbrmutants were selected by virtue of their resistance towardm-fluoro-d,l-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrAWT, but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrAfbrmutants were similar to those of the TyrAWTprotein. Analyses of the DNA sequences of thetyrAgenes revealed thattyrAfbrcontained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.
Paenibacillus polymyxa
Invades Plant Roots and Forms Biofilms
Authors: Timmusk Salme , Grantcharova Nina , Wagner E. Gerhart H.
Published: November 2005

Paenibacillus polymyxa
is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects
Arabidopsis thaliana
against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact.
12:
951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28,
in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa
, 2003). Here, we studied colonization of plant roots by a natural isolate of
P. polymyxa
which had been tagged with a plasmid-borne
gfp
gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.
Three Stages of a Biofilm Community Developing at the Liquid-Liquid Interface between Polychlorinated Biphenyls and Water
Authors: Macedo Alexandre José , Kuhlicke Ute , Neu Thomas R. , Timmis Kenneth N. , Abraham Wolf-Rainer
Published: November 2005

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera
Herbaspirillum
and
Bradyrhizobium
as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.
Enzymatic and Genetic Characterization of Carbon and Energy Metabolisms by Deep-Sea Hydrothermal Chemolithoautotrophic Isolates ofEpsilonproteobacteria
Authors: Takai Ken , Campbell Barbara J. , Cary S. Craig , Suzuki Masae , Oida Hanako , Nunoura Takuro , Hirayama Hisako , Nakagawa Satoshi , Suzuki Yohey , Inagaki Fumio , Horikoshi Koki
Published: November 2005

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophicEpsilonproteobacteriafrom deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All theEpsilonproteobacteriatested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities—ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase—while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genesaclB,porAB, andoorABand the lack of success at amplifying the form I and II RubisCO genes,cbbLandcbbM. The combination of enzymatic and genetic analyses demonstrates that theEpsilonproteobacteriatested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-seaEpsilonproteobacteriawere also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hynoperon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizingEpsilonproteobacteriashowed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5′-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-seaEpsilonproteobacteria.
Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists
Authors: Medina-Sánchez Juan M. , Felip Marisol , Casamayor Emilio O.
Published: November 2005

We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (
Bacteria
and
Archaea
) and, for the first time, to show archaeal cells ingested by mixotrophic protists.
Investigation of
Oscillatoria spongeliae
-Dominated Bacterial Communities in Four Dictyoceratid Sponges
Authors: Ridley Christian P. , John Faulkner D. , Haygood Margo G.
Published: November 2005

Certain species of marine sponges in the order
Dictyoceratida
harbor large populations of the cyanobacterial symbiont
Oscillatoria spongeliae
in the mesohyl (interior) of the sponge. We show that in four of these sponge species (
Lamellodysidea herbacea
,
Lamellodysidea chlorea
,
Lendenfeldia chondrodes
, and
Phyllospongia papyracea
) from Palau there is a consistent community of α-proteobacteria in addition to
O. spongeliae
that fall within the
Rhodobacter
group based on 16S rRNA gene analysis. Some of the α-proteobacteria in
Lendenfeldia chondrodes
and
P. papyracea
but not in the
Lamellodysidea
spp. contained site-specific insertions in the 16S rRNA gene. Reverse transcription-PCR experiments demonstrated that the largest insertion found in this study (63 bp) is present in the mature rRNA.
Lendenfeldia chondrodes
was the only sponge found to have another cyanobacterium in the tissue, a
Synechocystis
sp. We found that the
Synechocystis
sp. was present in both the pinacoderm (surface epithelial tissue) and mesohyl, in contrast to
O. spongeliae
, which was only found in the mesohyl through the use of specific fluorescence in situ hybridization experiments. Of the four sponge species, only
P. papyracea
was found to contain a significant number of γ-proteobacteria. These results demonstrate that
O. spongeliae
-dominated bacterial communities in different sponge species can vary considerably and increase our understanding of the bacterial communities found in marine invertebrates.
Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging toChloroflexiSubphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules
Authors: Yamada Takeshi , Sekiguchi Yuji , Imachi Hiroyuki , Kamagata Yoichi , Ohashi Akiyoshi , Harada Hideki
Published: November 2005

We previously reported that the thermophilic filamentous anaerobeAnaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylumChloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55°C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function ofAnaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties ofChloroflexisubphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze theChloroflexisubphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targetedChloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with theChloroflexisubphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using aChloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that theChloroflexisubphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to theChloroflexisubphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.
Design, Validation, and Application of a Seven-Strain
Staphylococcus aureus
PCR Product Microarray for Comparative Genomics
Authors: Witney Adam A. , Marsden Gemma L. , Holden Matthew T. G. , Stabler Richard A. , Husain Sarah E. , Vass J. Keith , Butcher Philip D. , Hinds Jason , Lindsay Jodi A.
Published: November 2005

Bacterial comparative genomics has been revolutionized by microarrays, but the power of any microarray is dependent on the number and diversity of gene reporters it contains.
Staphylococcus aureus
is an important human pathogen causing a wide range of invasive and toxin-mediated diseases, and more than 20% of the genome of any isolate consists of variable genes. Seven whole-genome sequences of
S. aureus
are available, and we exploited this rare opportunity to design, build, and validate a comprehensive, nonredundant PCR product microarray carrying reporters that represent every predicted open reading frame (3,623 probes). Such a comprehensive microarray necessitated a novel design strategy. Validation with the seven sequenced strains showed correct identification of 93.9% of genes present or absent/divergent but was dependent on the method of analysis chosen. Microarray data were highly reproducible, reducing the need for many replicate slides. Interpretation of microarray data was enhanced by focusing on the major areas of variation—the presence or absence of mobile genetic elements (MGEs). We compiled “composite genomes” of every individual MGE and visualized their distribution. This allowed the sensitive discrimination of related isolates, including the first clear description of how isolates of the same clone of epidemic methicillin-resistant
S. aureus
differ substantially in their carriage of MGEs. These MGEs carry virulence and resistance genes, suggesting differences in pathogenic potential. The novel methods of design and interpretation of data generated from this microarray will enable further studies of
S. aureus
evolution, epidemiology, and pathogenesis.
Sulfate-Reducing Bacteria in Floating Macrophyte Rhizospheres from an Amazonian Floodplain Lake in Bolivia and Their Association with Hg Methylation
Authors: Achá Darío , Iñiguez Volga , Roulet Marc , Guimarães Jean Remy Davée , Luna Ruddy , Alanoca Lucia , Sanchez Samanta
Published: November 2005

Five subgroups of sulfate-reducing bacteria (SRB) were detected by PCR in three macrophyte rhizospheres (
Polygonum densiflorum
,
Hymenachne donacifolia
, and
Ludwigia helminthorriza
) and three subgroups in
Eichhornia crassipes
from La Granja, a floodplain lake from the upper Madeira basin. The SRB community varied according to the macrophyte species but with different degrees of association with their roots. The rhizosphere of the C
4
plant
Polygonum densiflorum
had higher frequencies of SRB subgroups as well as higher mercury methylation potentials (27.5 to 36.1%) and carbon (16.06 ± 5.40%), nitrogen (2.03 ± 0.64%), Hg (94.50 ± 6.86 ng Hg g
−1
), and methylmercury (8.25 ± 1.45 ng Hg g
−1
) contents than the rhizosphere of the C
3
plant
Eichhornia crassipes
. Mercury methylation in
Polygonum densiflorum
and
Eichhornia crassipes
was reduced when SRB metabolism was inhibited by sodium molybdate.
On-Probe Sample Pretreatment for Direct Analysis of Lipids in Gram-Positive Bacterial Cells by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
Authors: Ishida Yasuyuki , Kitagawa Kuniyuki , Nakayama Akihito , Ohtani Hajime
Published: November 2005

On-probe sample pretreatment using trifluoroacetic acid as an additional reagent enabled the direct detection of phospholipids in whole bacteria by means of matrix-assisted laser desorption ionization mass spectrometry for not only gram-negative organisms but also gram-positive ones with a thicker peptidoglycan layer.
Cutinase-Like Enzyme from the Yeast
Cryptococcus
sp. Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics
Authors: Masaki Kazuo , Kamini Numbi Ramudu , Ikeda Hiroko , Iefuji Haruyuki
Published: November 2005

A purified lipase from the yeast
Cryptococcus
sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (ε-caprolactone), and poly(3-hydroxybutyrate).
Modeling Bacteriocin Resistance and Inactivation of
Listeria innocua
LMG 13568 by
Lactobacillus sakei
CTC 494 under Sausage Fermentation Conditions
Authors: Leroy Frédéric , Lievens Kristoff , De Vuyst Luc
Published: November 2005

In mixed cultures, bacteriocin production by the sausage isolate
Lactobacillus sakei
CTC 494 rapidly inactivated sensitive
Listeria innocua
LMG 13568 cells, even at low bacteriocin activity levels. A small fraction of the listerial population was bacteriocin resistant. However, sausage fermentation conditions inhibited regrowth of resistant cells.
Representational Difference Analysis and Real-Time PCR for Strain-Specific Quantification of
Lactobacillus sobrius
sp. nov
Authors: Konstantinov Sergey R. , Smidt Hauke , de Vos Willem M.
Published: November 2005

Lactobacillus sobrius
sp. nov., which was recently isolated from the intestine of weaning piglets, has potential probiotic properties. To follow the fate of
L. sobrius
strain 001
T
in dietary interventions, a novel and strain-specific quantitative detection procedure was developed. This procedure was based on the isolation of specific genomic fragments from the type strain by representational difference analysis and their detection by real-time PCR. The described strain-specific quantification approach may be used in studies aimed at tracking bacterial strains added to specific environments.
Heterogeneity of Putative Surface Layer Proteins in
Lactobacillus helveticus
Authors: Gatti Monica , Rossetti Lia , Fornasari Maria Emanuela , Lazzi Camilla , Giraffa Giorgio , Neviani Erasmo
Published: November 2005

The S-layer-encoding genes of 21
Lactobacillus helveticus
strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the
slpH1
gene of
L. helveticus
CNRZ 892 and a second cluster which includes genes similar to that of
prtY
. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species
L. helveticus
.
Metagenomic Analysis of the Microbial Community at Zodletone Spring (Oklahoma): Insights into the Genome of a Member of the Novel Candidate Division OD1
Authors: Elshahed Mostafa S. , Najar Fares Z. , Aycock Mandy , Qu Chunmei , Roe Bruce A. , Krumholz Lee R.
Published: November 2005

A metagenomic library was constructed from the anaerobic sediments of a mesophilic sulfur spring. Thirty-five bacterial 16S rRNA gene-containing clones were identified in this library. Analysis of a genomic fragment belonging to candidate division OD1 provided useful insights into the physiology and biochemistry of this novel, yet-uncultured candidate division.
Bacteriocin (Mutacin) Production by
Streptococcus mutans
Genome Sequence Reference Strain UA159: Elucidation of the Antimicrobial Repertoire by Genetic Dissection
Authors: Hale John D. F. , Ting Yi-Tian , Jack Ralph W. , Tagg John R. , Heng Nicholas C. K.
Published: November 2005

Streptococcus mutans
UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the
nlm
locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).
Trichosporon
Species Isolated from Guano Samples Obtained from Bat-Inhabited Caves in Japan
Authors: Sugita Takashi , Kikuchi Ken , Makimura Koichi , Urata Kensaku , Someya Takashi , Kamei Katsuhiko , Niimi Masakazu , Uehara Yoshimasa
Published: November 2005

Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of ∼700 yeast-like colonies, nine
Trichosporon
species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to
Trichosporon
species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that
Trichosporon
spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.
How to handle informed consent in longitudinal studies when participants have a limited understanding of the study
Authors: Helgesson G , Ludvigsson J , Gustafsson Stolt U
Published: November 2005

Empirical findings from a Swedish longitudinal screening study show that many of the research subjects had a limited understanding of the study. Nevertheless they were satisfied with the understanding they had and found it sufficient for informed continued participation. Were they wrong? In this paper, it is argued that the kind of understanding that is morally required depends partly on the kind of understanding on which the research subjects want to base their decisions, and partly on what kind of knowledge they lack. Researchers must ensure that the information process is not flawed and that participants receive the information they want. To achieve this, new information efforts may be needed. Researchers must also ensure that research subjects have knowledge about aspects of importance to them. Lack of understanding may, however, be the result of conscious choices by research subjects to disregard some of the information because it is not important to them. Such choices should normally be respected.
Epistemic paternalism in public health
Authors: Grill K , Hansson S O
Published: November 2005

Receiving information about threats to one’s health can contribute to anxiety and depression. In contemporary medical ethics there is considerable consensus that patient autonomy, or the patient’s right to know, in most cases outweighs these negative effects of information. Worry about the detrimental effects of information has, however, been voiced in relation to public health more generally. In particular, information aboutuncertainthreats to public health, from—for example, chemicals—are said to entail social costs that have not been given due consideration. This criticism implies a consequentialist argument for withholding such information from the public in their own best interest. In evaluating the argument for this kind of epistemic paternalism, the consequences of making information available must be compared to the consequences of withholding it. Consequences that should be considered include epistemic effects, psychological effects, effects on private decisions, and effects on political decisions. After giving due consideration to the possible uses of uncertain information and rebutting the claims that uncertainties imply small risks and that they are especially prone to entail misunderstandings and anxiety, it is concluded that there is a strong case against withholding of information about uncertain threats to public health.
Making sense of dignity
Authors: Ashcroft Richard E
Published: November 2005

In this review of Leon Kass’sLife, liberty and the defense of dignityand Deryck Beyleveld and Roger Brownsword’sHuman dignity in bioethics and biolaw. I consider the prospects for a theory of dignity as a basis for bioethics research. I argue that dignity theories are worth exploring in more detail, but that research needs to consider both “antitheory” accounts of the language of bioethics, and to give more weight to accounts of dignity as an outcome of holding positive liberties and as something that has a psychological dimension.
Ethical considerations in the application of preconditioning to solid organ transplantation
Authors: McNally S J , Harrison E M , Wigmore S J
Published: November 2005

The shortage of organs for transplantation has led researchers to look for new techniques to expand the donor pool. Preconditioning strategies have the potential to protect organs from transplant associated injury or may improve the function of substandard organs so that they become suitable for transplantation. Translating this type of technology to the clinical setting raises ethical issues, particularly relating to the deceased donor. It is important that society has the opportunity to discuss the issues raised by implementation of preconditioning strategies before they are implemented rather than as a reaction to them.
Human embryonic stem cell research debates: a Confucian argument
Authors: Tsai D F-C
Published: November 2005

Human embryonic stem cell research can bring about major biomedical breakthroughs and thus contribute enormously to human welfare, yet it raises serious moral problems because it involves using human embryos for experiment. The “moral status of the human embryo” remains the core of such debates. Three different positions regarding the moral status of the human embryo can be categorised: the“all”position, the“none”position, and the“gradualist”position.

The author proposes that the “gradualist” position is more plausible than the other two positions. Confucius’s moral principle ofjen, which proposes a unique theory of “love of gradation”, and the principle ofyi,which advocates “due treatment for persons”, are then explored. The author then argues that our moral obligations to do good to other living organisms, persons, and our families are different.

Putting together the “gradualist” position on the human embryo, and Confucius’s theories of “love of gradation” and “due treatment for persons”, the author concludes that the early embryo has less ethical significance than the later fetus and adult human. The moral obligation we have toward persons is clearer and stronger than that which we have toward human embryos. Embryo research is justifiable if it brings enormous welfare to human persons that cannot be otherwise achieved. The “love of gradation” requires us, however, to extend love and respect towards other entities according to their different status. We should therefore be very cautious in using human embryos for research, acknowledging the gradualist nature of their moral status.