bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Assessment of Picoeukaryotes in the Natural Environment by Using Taxon-Specific Oligonucleotide Probes in Association with Tyramide Signal Amplification-Fluorescence In Situ Hybridization and Flow Cytometry
Authors: Biegala Isabelle C. , Not Fabrice , Vaulot Daniel , Simon Nathalie 
Published: September 2003

ABSTRACT
Picoeukaryotes (cells of <3 μm in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3-μm-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.
History of the ADP/ATP-Translocase-Encoding Gene, a Parasitism Gene Transferred from a
Chlamydiales
Ancestor to Plants 1 Billion Years Ago
Authors: Greub Gilbert , Raoult Didier 
Published: September 2003

ABSTRACT

Nonmitochondrial ADP/ATP translocase is an energy parasite enzyme. Its encoding gene,
tlc
, is found only in
Rickettsiales
,
Chlamydiales
, and plant and alga plastids. We demonstrate the presence of
tlc
in
Parachlamydia acanthamoebae.
This gene shares more similarity with the
tlc
1 gene of
Chlamydiaceae
and the
tlc
of plant and alga plastids than with the
tlc
2 gene of
Chlamydiaceae
. Phylogenetic analysis, including all other
tlc
homologs found in GenBank, showed that
tlc
was duplicated in a
Chlamydiales
ancestor before the appearance of multicellular eukaryotes. A time scale, calibrated with seven independent time points obtained from fossil estimates and from the 16S rRNA molecular clock, was congruent with the molecular clock provided by
tlc
. Plant and alga plastids acquired
tlc
approximately when
Parachlamydiaceae
and
Chlamydiaceae
diverged, at the eucaryotic radiation time, ca. 1 billion years ago.
Plasmid Transfer from
Pseudomonas putida
to the Indigenous Bacteria on Alfalfa Sprouts: Characterization, Direct Quantification, and In Situ Location of Transconjugant Cells
Authors: Mølbak Lars , Licht Tine Rask , Kvist Thomas , Kroer Niels , Andersen Sigrid Rita 
Published: September 2003

ABSTRACT

The transfer of the plasmids pJKJ5 and TOL (pWWO) from
Pseudomonas putida
to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 × 10
−4
and 2.0 × 10
−6
transconjugant cells per donor cell for pKJK5::
gfp
and TOL::
gfp
, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera
Erwinia
and
Paenibacillus
were gradually replaced by
Pseudomonas
on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera
Pseudomonas
and
Erwinia.
The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from
P. putida
in the absence of selective pressure that could favor the presence of the investigated plasmids.
Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms
Authors: Lawrence J. R. , Swerhone G. D. W. , Leppard G. G. , Araki T. , Zhang X. , West M. M. , Hitchcock A. P. 
Published: September 2003

ABSTRACT
Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.
Foreshore Sand as a Source of
Escherichia coli
in Nearshore Water of a Lake Michigan Beach
Authors: Whitman Richard L. , Nevers Meredith B. 
Published: September 2003

ABSTRACT

Swimming advisories due to excessive
Escherichia coli
concentrations are common at 63rd Street Beach, Chicago, Ill. An intensive study was undertaken to characterize the source and fate of
E. coli
in beach water and sand at the beach. From April through September 2000, water and sand samples were collected daily or twice daily at two depths on three consecutive days per week (water samples,
n
= 1,747; sand samples,
n
= 858); hydrometeorological conditions and bird and bather distributions were also recorded.
E. coli
concentrations in sand and water were significantly correlated, with the highest concentration being found in foreshore sand, followed by those in submerged sediment and water of increasing depth. Gull contributions to
E. coli
densities in sand and water were most apparent on the day following gull activity in a given area.
E. coli
recolonized newly placed foreshore sand within 2 weeks. Analysis of variance, correlation, cluster analyses, concentration gradients, temporal-spatial distribution, demographic patterns, and DNA fingerprinting suggest that
E. coli
may be able to sustain population density in temperate beach sand during summer months without external inputs. This research presents evidence that foreshore beach sand (i) plays a major role in bacterial lake water quality, (ii) is an important non-point source of
E. coli
to lake water rather than a net sink, (iii) may be environmentally, and perhaps hygienically, problematic, and (iv) is possibly capable of supporting an autochthonous, high density of indicator bacteria for sustained periods, independent of lake, human, or animal input.
Differences in Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient
Authors: Feris Kevin , Ramsey Philip , Frazar Chris , Moore Johnnie N. , Gannon James E. , Holben William E. 
Published: September 2003

ABSTRACT
The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to α-, β-, and γ-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities.
Characterization of a Theta-Type Plasmid from
Lactobacillus sakei
: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli
Authors: Alpert Carl-Alfred , Crutz-Le Coq Anne-Marie , Malleret Christine , Zagorec Monique 
Published: September 2003

ABSTRACT

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from
Lactobacillus sakei
RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative
ori
segment containing 11- and 22-bp repeats and a
repA
gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an
Escherichia coli
replicon to produce the shuttle vector pRV566 and was observed to be functional in
L
.
sakei
for plasmid replication. The
L
.
sakei
replicon alone could not support replication in
E
.
coli
. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in
L
.
sakei
. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as
Lactobacillus curvatus
,
Lactobacillus casei
, and
Lactobacillus plantarum
, in addition to
L
.
sakei
, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.
A Gene Cluster for Chlorate Metabolism in
Ideonella dechloratans
Authors: Thorell Helena Danielsson , Stenklo Katarina , Karlsson Jan , Nilsson Thomas 
Published: September 2003

ABSTRACT

Chlorate reductase has been isolated from the chlorate-respiring bacterium
Ideonella dechloratans
, and the genes encoding the enzyme have been sequenced. The enzyme is composed of three different subunits and contains molybdopterin, iron, probably in iron-sulfur clusters, and heme
b
. The genes (
clr
) encoding chlorate reductase are arranged as
clrABDC
, where
clrA
,
clrB
, and
clrC
encode the subunits and
clrD
encodes a specific chaperone. Judging from the subunit composition, cofactor content, and sequence comparisons, chlorate reductase belongs to class II of the dimethyl sulfoxide reductase family. The
clr
genes are preceded by a novel insertion sequence (transposase gene surrounded by inverted repeats), denoted ISIde1. Further upstream, we find the previously characterized gene for chlorite dismutase (
cld
), oriented in the opposite direction. Chlorate metabolism in
I. dechloratans
starts with the reduction of chlorate, which is followed by the decomposition of the resulting chlorite to chloride and molecular oxygen. The present work reveals that the genes encoding the enzymes catalyzing both these reactions are in close proximity.
Wide Distribution of a Novel
pmoA
-Like Gene Copy among Type II Methanotrophs, and Its Expression in
Methylocystis
Strain SC2
Authors: Tchawa Yimga Merlin , Dunfield Peter F. , Ricke Peter , Heyer Jürgen , Liesack Werner 
Published: September 2003

ABSTRACT

Experiments were conducted to determine if a novel
pmoA
-like gene (
pmoA2
) recently discovered in the methane-oxidizing bacterium
Methylocystis
strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of
pmoA1
and
pmoA2
. By using this primer combination, we identified
pmoA2
in a wide range of type II MOB of the
Methylosinus-Methylocystis
group. However, screening by PCR and by Southern hybridization using a newly developed
pmoA2
-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor
pmoA2
. No
pmoA2
was detected in five type I MOB tested:
Methylococcus capsulatus
strain Bath,
Methylocaldum
strain E10A,
Methylobacter luteus
,
Methylomicrobium album
, and
Methylomonas
strain D1a. In comparative sequence analyses, all
pmoA2
-like sequences formed a coherent cluster clearly distinct from
pmoA1
sequences of type I and type II MOB, and from
amoA
sequences of the
Nitrosomonas
-
Nitrosospira
group. Phylogenetic analysis using the paml model suggested that
pmoA2
is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of
Methylocystis
strain SC2 for gene expression of
pmoA
. A strong signal was observed for
pmoA1
in Northern hybridization, while the results obtained for
pmoA2
were ambiguous. However, reverse transcription-PCR confirmed that
pmoA2
was expressed, albeit at lower level than
pmoA1
. This provided experimental evidence that the gene product of
pmoA2
may be a functionally active enzyme.
Isolation and Identification of Actinobacteria from Surface-Sterilized Wheat Roots
Authors: Coombs Justin T. , Franco Christopher M. M. 
Published: September 2003

ABSTRACT

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (
Triticum aestivum
L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including
Streptomyces
,
Microbispora
,
Micromonospora
, and
Nocardiodes
spp. Many of the
Streptomyces
spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to
Streptomyces
spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to
Streptomyces caviscabies
and
S. setonii
. None of these isolates, nor the
S. caviscabies
and
S. setonii
type strains, were found to carry the
nec1
pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.
Bacterial Diversity and Sulfur Cycling in a Mesophilic Sulfide-Rich Spring
Authors: Elshahed Mostafa S. , Senko John M. , Najar Fares Z. , Kenton Stephen M. , Roe Bruce A. , Dewers Thomas A. , Spear John R. , Krumholz Lee R. 
Published: September 2003

ABSTRACT

An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community. Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring. Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations. The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes. A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within δ-
Proteobacteria
, purple sulfur γ
-Proteobacteria
, ε
-Proteobacteria
,
Chloroflexi
, and filamentous
Cyanobacteria
of the order
Oscillatoria
as well as a novel group within γ
-Proteobacteria
. The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g.,
Cytophaga
,
Spirochaetes
,
Planctomycetes
,
Firmicutes
, and
Verrucomicrobiae
). Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments. In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions. These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions. The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene. DSR clones belonged to the
Desulfococcus
-
Desulfosarcina
-
Desulfonema
group,
Desulfobacter
group, and
Desulfovibrio
group as well as to a deeply branched group in the DSR tree with no representatives from cultures. Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats.
Purification and Properties of a Feruloyl Esterase Involved in Lignocellulose Degradation byAureobasidium pullulans
Authors: Rumbold Karl , Biely Peter , Mastihubová Maria , Gudelj Marinka , Gübitz Georg , Robra Karl-Heinz , Prior Bernard A. 
Published: September 2003

ABSTRACTThe lignocellulolytic fungusAureobasidium pullulansNRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methylp-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 μmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-Oand 5-Oarabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside,O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1,3)-O-β-d-xylopyranosyl-(1,4)-d-xylopyranose, andp-nitrophenyl-acetate but reveals only low activity onp-nitrophenyl-butyrate. The catalytic efficiency (kcat/Km) of the enzyme was highest on methylp-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.
Mutational Analysis of the Critical Bases Involved in Activation of the AreR-Regulated σ
54
-Dependent Promoter in
Acinetobacter
sp. Strain ADP1
Authors: Jones Rheinallt M. , Williams Peter A. 
Published: September 2003

ABSTRACT

The
areR
gene in
Acinetobacter
sp. strain ADP1 regulates the expression of the
areCBA
genes, which determine growth on benzyl alkanoates. AreR is a member of the NtrC/XylR family of regulatory proteins as determined by sequence homology. Seventy-nine bases upstream of the start of transcription is a region carrying two overlapping inverted repeat (IR) sequences that we predict to be the AreR binding site, also known as the upstream activator site (UAS). IR1 is a near-perfect (16 of 17 bp) repeat separated by 1 bp, and IR2 consists of 9- and 7-bp perfect repeats with a 3-bp gap, with the central bases of the two arms of the repeat separated by 44 and 22 bp. We report here a method for site-directed mutagenesis of chromosomal genes in ADP1 in which linear fragments generated by overlap extension PCR are used to transform ADP1 via its natural transformation system and recombinants are selected by a marker exchange-eviction strategy with a newly created
sacB
-Km cassette. This method was used to generate 38 strains with designed mutations in the putative UAS upstream of
areCBA.
The effects of the mutations on
areCBA
expression were measured by enzyme assays of benzyl alcohol dehydrogenase (AreB) and by reporter gene assays of
lacZ
inserted into
areA
. Substitutions or deletions in IR1 had more deleterious effects upon expression when they were in its central region, which overlaps the left arm of IR2, than when they were in its outer regions. By contrast, substitutions in the right arm of IR2 resulted in mutants with relatively high expression levels compared to that of the wild type. Effects of deletions in the right arm of IR2 were very dependent upon the length of the deletion, with 3- or 5-bp deletions reducing expression by >90% whereas an 11-bp deletion in the same area reduced the expression levels by only 50%, suggesting that alterations in the distance and the orientation of the UAS relative to the −24, −12 σ
54
promoter are critical.