bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Diversity of Rumen Methanogens from Sheep in Western Australia
Authors: Wright André-Denis G. , Williams Andrew J. , Winder Barbara , Christophersen Claus T. , Rodgers Sharon L. , Smith Kellie D. 
Published: March 2004

ABSTRACT

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order
Methanobacteriales
. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet.
Methanobrevibacter
strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.
Detection of
Arcobacter
spp. in the Coastal Environment of the Mediterranean Sea
Authors: Fera M. T. , Maugeri T. L. , Gugliandolo C. , Beninati C. , Giannone M. , La Camera E. , Carbone M. 
Published: March 2004

ABSTRACT

The occurrence of
Arcobacter
spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of
Arcobacter butzleri
,
Arcobacter cryaerophilus
, and
Arcobacter skirrowii
in cultures and environmental samples. Only one of the
Arcobacter
species,
A. butzleri
, was isolated from seawater and plankton samples. With some samples the
A. butzleri
PCR assay gave amplified products when cultures were negative.
A. cryaerophilus
and
A. skirrowii
were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of
Arcobacter
are present in marine environments. The assay was useful for detecting
Arcobacter
spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of
A. butzleri
and the presence of nonculturable
Arcobacter
spp. in the coastal environment of the Mediterranean Sea.
Molecular Diversity of New
Thermococcales
Isolates from a Single Area of Hydrothermal Deep-Sea Vents as Revealed by Randomly Amplified Polymorphic DNA Fingerprinting and 16S rRNA Gene Sequence Analysis
Authors: Lepage Elodie , Marguet Evelyne , Geslin Claire , Matte-Tailliez Oriane , Zillig Wolfram , Forterre Patrick , Tailliez Patrick 
Published: March 2004

ABSTRACT

Members of the
Thermococcales
are anaerobic
Archaea
belonging to the kingdom
Euryarchaea
that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new
Thermococcales
isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of
Thermococcus hydrothermalis
, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to
Pyrococcus
strains, whereas all the other isolates are
Thermococcus
strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of
Thermococcales
. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.
Regulatory Role of
Rhizobium etli
CNPAF512
fnrN
during Symbiosis
Authors: Moris Martine , Dombrecht Bruno , Xi Chuanwu , Vanderleyden Jos , Michiels Jan 
Published: March 2004

ABSTRACT

The
Rhizobium etli
CNPAF512
fnrN
gene was identified in the
fixABCX rpoN

2

region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of
R. etli fnrN
is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of
fnrN
is not controlled by the sigma factor RpoN and the symbiotic regulator
nifA
or
fixLJ
, but it is due to positive autoregulation. Inoculation of
Phaseolus vulgaris
with an
R. etli fnrN
mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the
R. etli fixN
,
fixG
, and
arcA
genes is strictly controlled by
fnrN
under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of
fixN
and
fixG
under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.
Novel Pathway of Salicylate Degradation by
Streptomyces
sp. Strain WA46
Authors: Ishiyama Daisuke , Vujaklija Dusica , Davies Julian 
Published: March 2004

ABSTRACT

A novel salicylate-degrading
Streptomyces
sp., strain WA46, was identified by UV fluorescence on solid minimal medium containing salicylate; trace amounts of gentisate were detected by high-pressure liquid chromatography when strain WA46 was grown with salicylate. PCR amplification of WA46 DNA with degenerate primers for gentisate 1,2-dioxygenase (GDO) genes produced an amplicon of the expected size. Sequential PCR with nested GDO primers was then used to identify a salicylate degradation gene cluster in a plasmid library of WA46 chromosomal DNA. The nucleotide sequence of a 13.5-kb insert in recombinant plasmid pWD1 (which was sufficient for the complete degradation of salicylate) showed that nine putative open reading frames (ORFs) (
sdgABCDEFGHR
) were involved. Plasmid pWD1 derivatives disrupted in each putative gene were transformed into
Streptomyces lividans
TK64. Disruption of either
sdgA
or
sdgC
blocked salicylate degradation; constructs lacking
sdgD
accumulated gentisate. Cell extracts from
Escherichia coli
DH5α transformants harboring pUC19 that expressed each of the
sdg
ORFs showed that conversions of salicylate to salicylyl-coenzyme A (CoA) and salicylyl-CoA to gentisyl-CoA required SdgA and SdgC, respectively. SdgA required CoA and ATP as cofactors, while NADH was required for SdgC activity; SdgC was identified as salicylyl-CoA 5-hydroxylase. Gentisyl-CoA underwent spontaneous cleavage to gentisate and CoA. SdgA behaved as a salicylyl-CoA ligase despite showing amino acid sequence similarity to an AMP-ligase. SdgD was identified as a GDO. These results suggest that
Streptomyces
sp. strain WA46 degrades salicylate by a novel pathway via a CoA derivative. Two-dimensional polyacrylamide gel electrophoresis and reverse transcriptase-PCR studies indicated that salicylate induced expression of the
sdg
cluster.
Physiology and Nutrition ofTreponema primitia, an H2/ CO2-Acetogenic Spirochete from Termite Hindguts
Authors: Graber Joseph R. , Breznak John A. 
Published: March 2004

ABSTRACTTreponema primitiastrains ZAS-1 and ZAS-2, the first spirochetes to be isolated from termite hindguts (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science283:686-689, 1999), were examined for nutritional, physiological, and biochemical properties relevant to growth and survival in their natural habitat. In addition to using H2plus CO2as substrates, these strains were capable of homoacetogenic growth on mono- and disaccharides and (in the case of ZAS-2) methoxylated benzenoids. Cells were also capable of mixotrophic growth (i.e., simultaneous utilization of H2and organic substrates). Cell extracts ofT. primitiapossessed enzyme activities of the Wood/Ljungdahl (acetyl coenzyme A) pathway of acetogenesis, including tetrahydrofolate-dependent enzymes of the methyl group-forming branch. However, a folate compound was required in the medium for growth. ZAS-1 and ZAS-2 growing on H2plus CO2displayed H2thresholds of 650 and 490 ppmv, respectively. Anoxic cultures of ZAS-1 and ZAS-2 maintained growth after the addition of as much as 0.5% (vol/vol) O2to the headspace atmosphere. Cell extracts exhibited NADH and NADPH peroxidase and NADH oxidase activities but neither catalase nor superoxide dismutase activity. Results indicate that (i)T. primitiais able to exploit a variety of substrates derived from the food of its termite hosts and in so doing contributes to termite nutrition via acetogenesis, (ii) in situ growth ofT. primitiais likely dependent on secretion of a folate compound(s) by other members of the gut microbiota, and (iii) cells possess enzymatic adaptations to oxidative stress, which is likely to be encountered in peripheral regions of the termite hindgut.
Description of
Treponema azotonutricium
sp. nov. and
Treponema primitia
sp. nov., the First Spirochetes Isolated from Termite Guts
Authors: Graber Joseph R. , Leadbetter Jared R. , Breznak John A. 
Published: March 2004

ABSTRACT

Long after their original discovery, termite gut spirochetes were recently isolated in pure culture for the first time. They revealed metabolic capabilities hitherto unknown in the
Spirochaetes
division of the
Bacteria
, i.e., H
2
plus CO
2
acetogenesis (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science
283:
686-689, 1999) and dinitrogen fixation (T. G. Lilburn, K. S. Kim, N. E. Ostrom, K. R. Byzek, J. R. Leadbetter, and J. A. Breznak, Science
292:
2495-2498, 2001). However, application of specific epithets to the strains isolated (
Treponema
strains ZAS-1, ZAS-2, and ZAS-9) was postponed pending a more complete characterization of their phenotypic properties. Here we describe the major properties of strain ZAS-9, which is readily distinguished from strains ZAS-1 and ZAS-2 by its shorter mean cell wavelength or body pitch (1.1 versus 2.3 μm), by its nonhomoacetogenic fermentation of carbohydrates to acetate, ethanol, H
2
, and CO
2
, and by 7 to 8% dissimilarity between its 16S rRNA sequence and those of ZAS-1 and ZAS-2. Strain ZAS-9 is proposed as the type strain of the new species,
Treponema azotonutricium
. Strains ZAS-1 and ZAS-2, which are H
2
-consuming, CO
2
-reducing homoacetogens, are proposed here to be two strains of the new species
Treponema primitia
. Apart from the salient differences mentioned above, the genomes of all three strains were similar in size (3,461 to 3,901 kb), in G+C content (50.0 to 51.0 mol%), and in possession of 2 copies of the gene encoding 16S rRNA (
rrs
). For comparison, the genome of the free-living spirochete
Spirochaeta aurantia
strain J1 was analyzed by the same methods and found to have a size of 3,719 kb, to contain 65.6 mol% G+C, and also to possess 2 copies of the
rrs
gene.
Characterization of the Arginine Deiminase Operon of
Streptococcus rattus
FA-1
Authors: Griswold Ann , Chen Yi-Ywan M. , Snyder Jennifer A. , Burne Robert A. 
Published: March 2004

ABSTRACT

The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the
arc
operon of
Streptococcus rattus
, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an
arcB
gene fragment obtained by degenerate PCRs, the FA-1
arc
operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in
S. rattus
FA-1 are organized as an
arcABCDT-adiR
operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The
arcA
transcriptional start site was identified by primer extension, and a σ
70
-like promoter was mapped 5′ to
arcA
. Reverse transcriptase PCR was used to establish that
arcABCDT
could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.
Wood-Destroying Soft Rot Fungi in the Historic Expedition Huts of Antarctica
Authors: Blanchette Robert A. , Held Benjamin W. , Jurgens Joel A. , McNew Douglas L. , Harrington Thomas C. , Duncan Shona M. , Farrell Roberta L. 
Published: March 2004

ABSTRACT

Three expedition huts in the Ross Sea region of Antarctica, built between 1901 and 1911 by Robert F. Scott and Ernest Shackleton, sheltered and stored the supplies for up to 48 men for 3 years during their explorations and scientific investigation in the South Pole region. The huts, built with wood taken to Antarctica by the early explorers, have deteriorated over the past decades. Although Antarctica has one of the coldest and driest environments on earth, microbes have colonized the wood and limited decay has occurred. Some wood in contact with the ground contained distinct microscopic cavities within secondary cell walls caused by soft rot fungi.
Cadophora
spp. could be cultured from decayed wood and other woods sampled from the huts and artifacts and were commonly associated with the soft rot attack. By using internal transcribed spacer sequences of ribosomal DNA and morphological characteristics, several species of
Cadophora
were identified, including
C. malorum
,
C. luteo-olivacea
, and
C. fastigiata
. Several previously undescribed
Cadophora
spp. also were found. At the Cape Evans and Cape Royds huts,
Cadophora
spp. commonly were isolated from wood in contact with the ground but were not always associated with soft rot decay. Pure cultures of
Cadophora
used in laboratory decay studies caused dark staining of all woods tested and extensive soft rot in
Betula
and
Populus
wood. The presence of
Cadophora
species, but only limited decay, suggests there is no immediate threat to the structural integrity of the huts. These fungi, however, are widely found in wood from the historic huts and have the capacity to cause extensive soft rot if conditions that are more conducive to decay become common.
Heat Shock Response in
Lactobacillus plantarum
Authors: De Angelis Maria , Di Cagno Raffaella , Huet Claude , Crecchio Carmine , Fox Patrick F. , Gobbetti Marco 
Published: March 2004

ABSTRACT

Heat stress resistance and response were studied in strains of
Lactobacillus plantarum
. Stationary-phase cells of
L. plantarum
DPC2739 had decimal reduction times (
D
values) (
D
value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75°C, respectively. When mid-exponential-phase cells were used, the
D
values decreased. The temperature increases which caused a 10-fold reduction in the
D
value ranged from 9 to 20°C, depending on the strain. Part of the cell population treated at 72°C for 90 s recovered viability during incubation at 7°C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of
L. plantarum
DPC2739 were adapted to 42°C for 1 h, the heat resistance at 72°C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of
L. plantarum
DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of
L. plantarum
is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in
L. plantarum
are fundamental for survival in cheese during manufacture.
Molecular Detection and Identification ofBrettanomyces/Dekkera bruxellensisandBrettanomyces/Dekkera anomalusin Spoiled Wines
Authors: Cocolin Luca , Rantsiou Kalliopi , Iacumin Lucilla , Zironi Roberto , Comi Giuseppe 
Published: March 2004

ABSTRACTIn this paper we describe the development of a PCR protocol to specifically detectBrettanomyces bruxellensisandB. anomalus.Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from otherBrettanomycesspp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis withDdeI to differentiate betweenB. bruxellensisandB. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection ofB. bruxellensisandB. anomalusin wines suspected to be spoiled byBrettanomycesspp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled byB. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.
Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in
Ceratocystis ulmi
Authors: Hornby Jacob M. , Jacobitz-Kizzier Sarah M. , McNeel Donna J. , Jensen Ellen C. , Treves David S. , Nickerson Kenneth W. 
Published: March 2004

ABSTRACT

We studied the inoculum size effect in
Ceratocystis ulmi
, the dimorphic fungus that causes Dutch elm disease. In a defined glucose-proline-salts medium, cells develop as budding yeasts when inoculated at ≥10
6
spores per ml and as mycelia when inoculated at <10
6
spores per ml. The inoculum size effect was not influenced by inoculum spore type, age of the spores, temperature, pH, oxygen availability, trace metals, sulfur source, phosphorous source, or the concentration of glucose or proline. Similarly, it was not influenced by added adenosine, reducing agents, methyl donors, amino sugars, fatty acids, or carbon dioxide. Instead, growing cells excreted an unknown quorum-sensing factor that caused a morphological shift from mycelia to budding yeasts. This yeast-promoting effect is abolished if it is extracted with an organic solvent such as ethyl acetate. The quorum-sensing activity acquired by the organic solvent could be added back to fresh medium in a dose-dependent fashion. The quorum-sensing activity in
C. ulmi
spent medium was specific for
C. ulmi
and had no effect on the dimorphic fungus
Candida albicans
or the photomorphogenic fungus
Penicillium isariaeforme
. In addition, farnesol, the quorum-sensing molecule produced by
C. albicans
, did not inhibit mycelial development of
C. ulmi
when present at concentrations of up to 100 μM. We conclude that the inoculum size effect is a manifestation of a quorum-sensing system that is mediated by an excreted extracellular molecule, and we suggest that quorum sensing is a general phenomenon in dimorphic fungi.
Development and Use of Flow Cytometry for Detection of Airborne Fungi
Authors: Prigione Valeria , Lingua Guido , Marchisio Valeria Filipello 
Published: March 2004

ABSTRACT

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of
Aspergillus fumigatus
and
Penicillium brevicompactum
conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.