bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metagenome Survey of Biofilms in Drinking-WaterNetworks
Authors: Schmeisser C. , Stöckigt C. , Raasch C. , Wingender J. , Timmis K. N. , Wenderoth D. F. , Flemming H.-C. , Liesegang H. , Schmitz R. A. , Jaeger K.-E. , Streit W. R. 
Published: December 2003

ABSTRACT

Most
naturally occurring biofilms contain a vast majority of microorganisms
which have not yet been cultured, and therefore we have little
information on the genetic information content of these communities.
Therefore, we initiated work to characterize the complex metagenome of
model drinking water biofilms grown on rubber-coated valves by
employing three different strategies. First, a sequence analysis of 650
16S rRNA clones indicated a high diversity within the biofilm
communities, with the majority of the microbes being closely related to
the
Proteobacteria
. Only a small fraction of the 16S rRNA
sequences were highly similar to rRNA sequences from
Actinobacteria
, low-G+C gram-positives and the
Cytophaga
-
Flavobacterium
-
Bacteroides
group.
Our second strategy included a snapshot genome sequencing approach.
Homology searches in public databases with 5,000 random sequence clones
from a small insert library resulted in the identification of 2,200
putative protein-coding sequences, of which 1,026 could be classified
into functional groups. Similarity analyses indicated that significant
fractions of the genes and proteins identified were highly similar to
known proteins observed in the genera
Rhizobium
,
Pseudomonas
, and
Escherichia
. Finally, we report 144
kb of DNA sequence information from four selected cosmid clones, of
which two formed a 75-kb overlapping contig. The majority of the
proteins identified by whole-cosmid sequencing probably originated from
microbes closely related to the alpha-, beta-, and
gamma-
Proteobacteria.
The sequence information was used to set
up a database containing the phylogenetic and genomic information on
this model microbial community. Concerning the potential health risk of
the microbial community studied, no DNA or protein sequences directly
linked to pathogenic traits were
identified.
Seasonal Changes in the Rhizosphere MicrobialCommunities Associated with Field-Grown Genetically ModifiedCanola (
Brassicanapus
)
Authors: Dunfield Kari E. , Germida James J. 
Published: December 2003

ABSTRACT
The
introduction of transgenic plants into agricultural ecosystems has
raised the question of the ecological impact of these plants on
nontarget organisms, such as soil bacteria. Although differences in
both the genetic structure and the metabolic function of the microbial
communities associated with some transgenic plant lines have been
established, it remains to be seen whether these differences have an
ecological impact on the soil microbial communities. We conducted a
2-year, multiple-site field study in which rhizosphere samples
associated with a transgenic canola variety and a conventional canola
variety were sampled at six times throughout the growing season. The
objectives of this study were to identify differences between the
rhizosphere microbial community associated with the transgenic plants
and the rhizosphere microbial community associated with the
conventional canola plants and to determine whether the differences
were permanent or depended on the presence of the plant.
Community-level physiological profiles, fatty acid methyl ester
profiles, and terminal amplified ribosomal DNA restriction analysis
profiles of rhizosphere microbial communities were compared to the
profiles of the microbial community associated with an unplanted,
fallow field plot. Principal-component analysis showed that there was
variation in the microbial community associated with both canola
variety and growth season. Importantly, while differences between the
microbial communities associated with the transgenic plant variety were
observed at several times throughout the growing season, all analyses
indicated that when the microbial communities were assessed after
winter, there were no differences between microbial communities from
field plots that contained harvested transgenic canola plants and
microbial communities from field plots that did not contain plants
during the field season. Hence, the changes in the microbial community
structure associated with genetically modified plants were temporary
and did not persist into the next field
season.
Use of a Green Fluorescent Strain for Analysis ofXylella fastidiosaColonization ofVitis vinifera
Authors: Newman Karyn L. , Almeida Rodrigo P. P. , Purcell Alexander H. , Lindow Steven E. 
Published: December 2003

ABSTRACTXylella fastidiosacauses Pierce's disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants.X. fastidiosacolonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineeredX. fastidiosato express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host,Vitis vinifera.In symptomatic leaves, the fraction of vessels colonized byX. fastidiosawas fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked byX. fastidiosacolonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence forX. fastidiosamovement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.
In Vitro andIn Vivo Studies of the Yrp1 Protease from
Yersinia ruckeri
andIts Role in Protective Immunity against Enteric Red MouthDisease ofSalmonids
Authors: Fernandez L. , Lopez J. R. , Secades P. , Menendez A. , Marquez I. , Guijarro J. A. 
Published: December 2003

ABSTRACT

Yersinia
ruckeri
, the etiological agent of the enteric red mouth disease
(ERM) of salmonids, produces Yrp1, a serralysin metalloprotease
involved in pathogenesis. We describe here the hydrolytic and
immunogenic properties of Yrp1. The protease was able to hydrolyze
different matrix and muscle proteins as laminin, fibrinogen,
gelatine, actin, and myosin but not type II and IV
collagens. In addition, the Yrp1 protein, when inactivated by heat and
used as an immunogen, was able to elicit a strong protection against
the development of ERM. The analysis of different
Y. ruckeri
strains with (Azo
+
) or without
(Azo
−
) Yrp1 activity showed that all of them
contained the
yrp1
operon. By using
yrp1
::
lacZ
operon fusions,
protease production analysis, and complementation studies, it was
possible to show that an Azo
−
strain was blocked at
the transcription level. The transcriptional study of the
yrp1
operon under different environmental conditions showed that it was
regulated by osmolarity and temperature, without pH influence. Finally,
when β-galactosidase activity was used as a probe in vivo, the
progression of the disease in the fish could be visualized, and the
tropism of the bacterium and affected organs could be defined. This
system opens a vast field of study not only with regard to fish disease
progression but also in pathogen interactions, temporal gene
expression, carrier stages, antibiotic resistance selection,
etc.
Concomitant Induction of Systemic Resistance to
Pseudomonas syringae
pv.
lachrymans
in Cucumber by
Trichoderma asperellum
(T-203) and Accumulation ofPhytoalexins
Authors: Yedidia Iris , Shoresh Michal , Kerem Zohar , Benhamou Nicole , Kapulnik Yoram , Chet Ilan 
Published: December 2003

ABSTRACT

Most
studies on the reduction of disease incidence in soil treated with
Trichoderma asperellum
have focused on microbial interactions
rather than on plant responses. This study presents conclusive evidence
for the induction of a systemic response against angular leaf spot of
cucumber (
Pseudomonas syringae
pv.
lachrymans
)
following application of
T. asperellum
to the root system. To
ascertain that
T. asperellum
was the only microorganism
present in the root milieu, plants were grown in an aseptic hydroponic
growth system. Disease symptoms were reduced by as much as 80%,
corresponding to a reduction of 2 orders of magnitude in bacterial cell
densities in leaves of plants pretreated with
T. asperellum
.
As revealed by electron microscopy, bacterial cell proliferation in
these plants was halted. The protection afforded by the biocontrol
agent was associated with the accumulation of mRNA of two defense
genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia
lyase (
PAL
) and the lipoxygenase pathway gene encoding
hydroxyperoxide lyase (
HPL
). This was further supported by the
accumulation of secondary metabolites of a phenolic nature that showed
an increase of up to sixfold in inhibition capacity of bacterial growth
in vitro. The bulk of the antimicrobial activity was found in the
acid-hydrolyzed extract containing the phenolics in their aglycone
form. High-performance liquid chromatography analysis of
phenolic compounds showed a marked change in their profile in the
challenged, preelicited plants relative to that in challenged
controls. The results suggest that similar to beneficial
rhizobacteria,
T. asperellum
may activate separate metabolic
pathways in cucumber that are involved in plant signaling and
biosynthesis, eventually leading to the systemic accumulation of
phytoalexins.
Molecular Evidence for Novel Planctomycete Diversityin a Municipal Wastewater TreatmentPlant
Authors: Chouari Rakia , Le Paslier Denis , Daegelen Patrick , Ginestet Philippe , Weissenbach Jean , Sghir Abdelghani 
Published: December 2003

ABSTRACT

We
examined anoxic and aerobic basins and an anaerobic digestor of a
municipal wastewater treatment plant for the presence of novel
planctomycete-like diversity. Three 16S rRNA gene libraries were
constructed by using a 16S rRNA-targeted universal reverse primer and a
forward PCR primer specific for
Planctomycetes
. Phylogenetic
analysis of 234 16S rRNA gene sequences defined 110 operational
taxonomic units. The majority of these sequences clustered with the
four known genera,
Pirellula
(32%),
Planctomyces
(18.4%),
Gemmata
(3.8%),
and
Isosphaera
(0.4%). More interestingly, 42.3%
of the sequences appeared to define two distantly separated
monophyletic groups. The first group, represented by 35.5% of
the sequences, was related to the
Planctomyces
group and
branched as a monophyletic cluster. It exhibited between 11.9 and
20.3% 16S rRNA gene sequence dissimilarity in comparisons with
cultivated planctomycetes. The second group, represented by 6.8%
of the sequences, was deeply rooted within the
Planctomycetales
tree. It was distantly related to the anammox
sequences (level of dissimilarity, 20.3 to 24.4%) and was a
monophyletic cluster. The retrieved sequences extended the
intralineage phylogenetic depth of the
Plantomycetales
from 23
to 30.6%. The lineages described here may have a broad diversity
of undiscovered biochemical and metabolic novelty. We
developed a new 16S rRNA-targeted oligonucleotide probe and localized
members of one of the phylogenetic groups using the fluorescent in situ
hybridization technique. Our results indicate that activated sludge
contains very diverse representatives of this group, which grow under
aerobic and anoxic conditions and even under anaerobic conditions. The
majority of species in this group remain poorly
characterized.
Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of
Streptococcus pneumoniae
Strain TIGR4
Authors: Trzcinski Krzysztof , Thompson Claudette M. , Lipsitch Marc 
Published: December 2003

ABSTRACT

The
polysaccharide capsule is the primary virulence factor in
Streptococcus pneumoniae
. There are at least 90 serotypes of
S. pneumoniae
, identified based on the immunogenicity of
different capsular sugars. The aim of this study was to construct
pneumococcal strains that are isogenic except for capsular type.
Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational
replacement of the capsular polysaccharide synthesis (
cps
)
locus with the bicistronic Janus cassette (C. K. Sung,
J. P. Claverys, and D. A. Morrison, Appl. Environ.
Microbiol. 67:5190-5196, 2001). In subsequent transformation with
chromosomal DNA, the cassette was replaced by the
cps
locus
derived from a strain of a different serotype, either 6B, 7F, 14, or
19F. To minimize the risk of uncontrolled recombinational replacements
in loci other than
cps
, the
TIGR
cps
::Janus strain was“
backcross” transformed three times with chromosomal
DNA of subsequently constructed capsular type transformants. Capsular
serotypes were confirmed in all new capsule variants by the Quellung
reaction. Restriction fragment length polymorphism (RFLP) analysis of
the
cps
locus confirmed the integrity of the
cps
region transformed into the TIGR strain, and RFLP of the flanking
regions confirmed their identities with the corresponding regions of
the recipient. Transformants had in vitro growth rates greater than or
equal to that of TIGR4. All four strains were able to colonize C57BL/6
mice (female, 6 weeks old) for at least 7 days when mice were
intranasally inoculated with 6 × 10
6
to 8 ×
10
6
CFU. The constructed capsular variants of TIGR4 are
suitable for use in studies on the role of
S. pneumoniae
capsular polysaccharide in immunity, colonization, and
pathogenesis.
Effects ofSaxitoxin (STX) and Veratridine on BacterialNa
+
-K
+
Fluxes: a Prokaryote-BasedSTXBioassay
Authors: Pomati Francesco , Rossetti Carlo , Calamari Davide , Neilan Brett A. 
Published: December 2003

ABSTRACT

Saxitoxin
(STX) is a potent natural sodium channel blocker and represents a
significant health concern worldwide. We describe here the antagonistic
effects of STX and veratridine (VTD), an Na
+
channel
activator, on three gram-negative bacteria and their application to an
STX bioassay. STX reduced the total cellular levels of both
Na
+
and K
+
, as measured by flame
photometry, whereas VTD increased the cellular concentrations relative
to control ion fluxes in the cyanobacterium
Cylindrospermopsis
raciborskii
AWT205. Endogenous STX production in toxic
cyanobacterial strains of
C. raciborskii
and
Anabaena
circinalis
prevented cell lysis induced by VTD stress. Microscopic
cell counts showed that non-STX producing cyanobacteria displayed
complete cell lysis and trichome fragmentation 5 to 8 h after
addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The
addition of STX, or its analogue neoSTX, prior to treatment with VTD
plus VAN prevented complete lysis in non-STX-producing cyanobacteria.
VTD also affected cyanobacterial metabolism, and the presence of
exogenous STX in the sample also ameliorated this decrease in metabolic
activity, as measured by the cellular conversion of tetrazolium into
formazan. Reduced primary metabolism was also recorded as a decrease in
the light emissions of
Vibrio fischeri
exposed to VTD.
Addition of STX prior to VTD resulted in a rapid and dose-dependent
response to the presence of the channel blocker, with samples
exhibiting resistance to the VTD effect. Our findings demonstrate that
STX and VTD influence bacterial Na
+
and
K
+
fluxes in opposite ways, and these
principles can be applied to the development of a prokaryote-based STX
bioassay.
Detection of Infectious Adenovirus in Cell Culture bymRNA ReverseTranscription-PCR
Authors: Ko Gwangpyo , Cromeans Theresa L. , Sobsey Mark D. 
Published: December 2003

ABSTRACT

We
have developed and evaluated the reverse transcription (RT)-PCR
detection of mRNA in cell culture to assay infectious adenoviruses
(Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads
are detected because they are the only ones able to produce mRNA during
replication in cell culture. Three primer sets for RT-PCR amplification
of mRNA were evaluated for their sensitivity and specificity: a
conserved region of late mRNA transcript encoding a virion structural
hexon protein and detecting a wide range of human Ads and two primer
sets targeting a region of an early mRNA transcript that specifically
detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549
and Graham 293 cells were recovered from cell lysates with oligo(dT) at
different time periods after infection and treated with RNase-free
DNase to remove residual contaminating DNA, and then Ad mRNA was
detected by RT-PCR assay. The mRNA of Ad2 was detected as early as
6 h after infection at 10
6
infectious units (IU)
per cell culture and after longer incubation times at levels as low as
1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as
24 h after infection at 10
6
IU per cell culture
and at levels as low as 5 IU per cell culture after longer incubation
times. To confirm the detection of only infectious viruses, it was
shown that no mRNA was detected from Ad2 and Ad41 inactivated by free
chlorine or high doses of collimated, monochromatic (254-nm) UV
radiation. Detection of Ad2 mRNA exactly coincided with the presence of
virus infectivity detected by cytopathogenic effects in cell cultures,
but mRNA detection occurred sooner. These results suggest that mRNA
detection by RT-PCR assay in inoculated cell cultures is a very
sensitive, specific, and rapid method by which to detect infectious Ads
in water and other environmental
samples.
Cometabolism ofMethyl
tertiary
Butyl Ether and Gaseous
n
-Alkanes by
Pseudomonas mendocina
KR-1 Grown on C
5
toC
8
n
-Alkanes
Authors: Smith Christy A. , O'Reilly Kirk T. , Hyman Michael R. 
Published: December 2003

ABSTRACT

Pseudomonas
mendocina
KR-1 grew well on toluene,
n
-alkanes
(C
5
to C
8
), and 1° alcohols
(C
2
to C
8
) but not on other aromatics, gaseous
n
-alkanes (C
1
to C
4
), isoalkanes
(C
4
to C
6
), 2° alcohols (C
3
to
C
8
), methyl
tertiary
butyl ether (MTBE), or
tertiary
butyl alcohol (TBA). Cells grown under carbon-limited
conditions on
n
-alkanes in the presence of MTBE (42μ
mol) oxidized up to 94% of the added MTBE to TBA. Less
than 3% of the added MTBE was oxidized to TBA when cells were
grown on either 1° alcohols, toluene, or dextrose in the
presence of MTBE. Concentrated
n
-pentane-grown cells oxidized
MTBE to TBA without a lag phase and without generating
tertiary
butyl formate (TBF) as an intermediate. Neither TBF
nor TBA was consumed by
n
-pentane-grown cells, while
formaldehyde, the expected C
1
product of MTBE dealkylation,
was rapidly consumed. Similar

K
s

values for MTBE
were observed for cells grown on C
5
to C
8
n
-alkanes (12.95 ± 2.04 mM), suggesting that the same
enzyme oxidizes MTBE in cells grown on each
n
-alkane. All
growth-supporting
n
-alkanes (C
5
to C
8
)
inhibited MTBE oxidation by resting
n
-pentane-grown cells.
Propane (

K
i

= 53 μM) and
n
-butane (

K
i

= 16 μM) also
inhibited MTBE oxidation, and both gases were also consumed by cells
during growth on
n
-pentane. Cultures grown on C
5
to
C
8
n
-alkanes also exhibited up to twofold-higher
levels of growth in the presence of propane or
n
-butane,
whereas no growth stimulation was observed with methane, ethane, MTBE,
TBA, or formaldehyde. The results are discussed in terms of their
impacts on our understanding of MTBE biodegradation and
cometabolism.
Application of a Specific and Sensitive RadiometricAssay for Microbial Lipase Activities in Marine Water Samplesfrom the Lagoon ofNouméa
Authors: Bourguet Nicolas , Torréton Jean-Pascal , Galy Olivier , Arondel Vincent , Goutx Madeleine 
Published: December 2003

ABSTRACT

Marine
microbiologists commonly assay lipase activities by using a synthetic
fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The
technique is convenient, but it is considered to be unspecific because
of the structure of this analog. This study reports the design of a new
specific and sensitive lipase assay based on the use of a radiolabeled
triglyceride, [
3
H]triolein. Free fatty acids (FFA)
resulting from its hydrolysis are isolated as a function of time in a
one-step liquid-liquid extraction and then radioassayed. MUF-oleate and
[
3
H]triolein techniques were compared by measuring
lipase activities at similar substrate concentrations along a trophic
gradient in the Southwest Lagoon of New Caledonia, near Nouméa.
Hydrolysis rates decreased from the nearshore station to the offshore
station and showed similar trends regardless of the technique used.
Rates decreased from 5.83 to 0.88 nmol of
FFA · liter
−1
· h
−1
and from 0.76 to 0.23 nmol of
3
H-FFA · liter
−1
· h
−1
,
respectively. These results appeared to be consistent with bacterial
production results, which also decreased similarly (from 0.59 to 0.26μ
g of
C · liter
−1
· h
−1
).
However, the ratio of MUF-oleate activities to
[
3
H]triolein activities, which was constant at the
offshore stations (3.8 ± 0.1), gradually increased
at the nearshore stations (from 4.1 to 7.6). This result shows that the
two assays respond in different ways to changes in environmental
conditions and validates the need to set up more specific enzymatic
assays.
Transporter-MediatedUptake of 2-Chloro- and 2-Hydroxybenzoate by
Pseudomonashuttiensis
StrainD1
Authors: Yuroff A. S. , Sabat G. , Hickey W. J. 
Published: December 2003

ABSTRACT

We
investigated the mechanisms of uptake of 2-chlorobenzoate (2-CBa) and
2-hydroxybenzoate (2-HBa) by
Pseudomonas huttiensis
strain D1.
Uptake was monitored by assaying intracellular accumulation of
2-[UL-
ring
-
14
C]CBa and
2-[UL-
ring
-
14
C]HBa. Uptake of 2-CBa
showed substrate saturation kinetics with an apparent

K
m

of 12.7 ± 2.6 μM and a maximum
velocity (
V
max
) of 9.76 ± 0.78 nmol
min
−1
mg of protein
−1
. Enhanced
rates of uptake were induced by growth on 2-CBa and 2-HBa, but not by
growth on benzoate or 2,5-di-CBa. Intracellular accumulations of 2-CBa
and 2-HBa were 109- and 42-fold greater, respectively, than the
extracellular concentrations of these substrates and were indicative of
uptake mediated by a transporter rather than driven by substrate
catabolism (“metabolic drag”). Results of competitor
screening tests indicated that the substrate range of the transporter
did not include other
o
-halobenzoates that serve as growth
substrates for strain D1 and for which the metabolism was initiated by
the same dioxygenase as 2-CBa and 2-HBa. This suggested that multiple
mechanisms for substrate uptake were coupled to the same catabolic
enzyme. The preponderance of evidence from tests with metabolic
inhibitors and artificial electrochemical gradients suggested that
2-CBa uptake was driven by ATP hydrolysis. If so, the 2-CBa transporter
would be the first of the ATP binding cassette type implicated in
uptake of haloaromatic
acids.