bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Real-Time PCR for Determination ofMicrocystin Synthetase E Copy Numbers forMicrocystisandAnabaenainLakes
Authors: Vaitomaa Jaana , Rantala Anne , Halinen Katrianna , Rouhiainen Leo , Tallberg Petra , Mokelke Lena , Sivonen Kaarina
Published: December 2003

Cyanobacterial mass occurrences in freshwater lakes are generally formed byAnabaena,Microcystis, andPlanktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum ofMicrocystisandAnabaena mcyEcopy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating thatmcyEgene copy numbers can be used as surrogates for hepatotoxicMicrocystisandAnabaena. The main microcystin producer in Lake Tuusulanjärvi wasMicrocystisspp., since averageMicrocystis mcyEcopy numbers were >30 times more abundant than those ofAnabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxicAnabaenaas well as toxicMicrocystisstrains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.
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

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

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

Xylella 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

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

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

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

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

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

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

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

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

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.
Genetic Diversity of
Campylobacter jejuni
Isolates from Farm Animals and the FarmEnvironment
Authors: Colles F. M. , Jones K. , Harding R. M. , Maiden M. C. J.
Published: December 2003

The
genetic diversity of
Campylobacter jejuni
isolates from farm
animals and their environment was investigated by multilocus sequence
typing (MLST). A total of 30 genotypes, defined by allelic profiles
(assigned to sequence types [STs]), were found in 112
C.
jejuni
isolates originating in poultry, cattle, sheep, starlings,
and slurry. All but two of these genotypes belonged to one of nine
C. jejuni
clonal complexes previously identified in isolates
from human disease and retail food samples and one clonal complex
previously associated with an environmental source. There was some
evidence for the association of certain clonal complexes with
particular farm animals: isolates belonging to the ST-45 complex
predominated among poultry isolates but were absent among sheep
isolates, while isolates belonging to the ST-61 and ST-42 complexes
were predominant among sheep isolates but were absent from the poultry
isolates. In contrast, ST-21 complex isolates were distributed among
the different isolation sources. Comparison with MLST data from 91
human disease isolates showed small but significant genetic
differentiation between the farm and human isolates; however,
representatives of six clonal complexes were found in both samples.
These data demonstrate that MLST and the clonal complex model can be
used to identify and compare the genotypes of
C. jejuni
isolates from farm animals and the environment with those from retail
food and human
disease.
Predominance of
vanA
Genotype amongVancomycin-Resistant
Enterococcus
Isolates from Poultry andSwine in CostaRica
Authors: Bustamante Warner , Alpízar Angélica , Hernández Silvia , Pacheco Alexandra , Vargas Natalia , Herrera Marco Luis , Vargas Álvaro , Caballero Magaly , García Fernando
Published: December 2003

The
use of avoparcin as a growth promoter is considered to have selected
for vancomycin-resistant enterococci (VRE). In Costa Rica, the use of
avoparcin for poultry and swine was intensive until the product was
withdrawn from the market in 2000. We evaluated the presence of VRE in
poultry, swine, and cattle fecal samples obtained during 1998 and 1999.
A total of 185 VRE isolates were recovered from 116 out of 893 samples.
Enterococcus faecium
was the most frequently isolated species
(50.8%), being predominant among poultry (71.6%) and
swine (37.7%) isolates, but it was not recovered from the bovine
samples. The second-most-frequently-isolated species from poultry and
swine, respectively, were
E. durans
(23.2%)
and
E. faecalis
(21.7%).
E. casseliflavus
was the only species obtained from bovine
samples, but it was not found among the avian isolates. An evident
predominance of the
vanA
determinant among
vancomycin-resistant enterococcal species from poultry and swine, but
not from cattle, was observed and was similar to the situation in
European countries before avoparcin was forbidden. The diversity of the
vanA
determinant in the isolates was assessed by detection of
the IS
1251
insertion in the
vanSH
intergenic region
and of the IS
1476
insertion in the
vanXY
intergenic
region. However, in none of the 154
vanA
+
isolates recovered in this study were those insertions
detected.
Spatial Analysis of Archaeal Community Structure inGrasslandSoil
Authors: Nicol Graeme W. , Glover L. Anne , Prosser James I.
Published: December 2003

The
complex structure of soil and the heterogeneity of resources available
to microorganisms have implications for sampling regimens when the
structure and diversity of microbial communities are analyzed. To
assess the heterogeneity in community structure, archaeal communities,
which typically contain sequences belonging to the nonthermophilic
Crenarchaeota
, were examined at two contrasting spatial scales
by using PCR-denaturing gradient gel electrophoresis (DGGE) analysis
followed by unweighted pair group method with arithmetic mean analysis
of 16S rRNA- and ribosomal DNA-derived profiles. A
macroscale analysis was carried out with soil cores taken
at 2-m intervals along triplicate 8-m transects from both managed
(improved) and natural (unimproved) grassland rhizosphere soils. A
microscale analysis was carried out with a single soil core by
assessing the effects of both sample size (10, 1, and 0.1 g)
and distance between samples. The much reduced complexity of archaeal
profiles compared to the complexity typical of the bacterial community
facilitated visual comparison of profiles based on band presence and
revealed different levels of heterogeneity between sets of samples. At
the macroscale level, heterogeneity over the transect could not be
related to grassland type. Substantial heterogeneity was observed
across both improved and unimproved transects, except for one improved
transect that exhibited substantial homogeneity, so that profiles for a
single core were largely representative of the entire transect. At the
smaller scale, the heterogeneity of the archaeal community structure
varied with sample size within a single 8- by 8-cm core. The archaeal
DGGE profiles for replicate 10-g soil samples were similar, while those
for 1-g samples and 0.1-g samples showed greater heterogeneity. In
addition, there was no relationship between the archaeal profiles and
the distance between 1- or 0.1-g samples, although relationships
between community structure and distance of separation may occur at a
smaller scale. Our findings demonstrate the care required when workers
attempt to obtain a representative picture of microbial community
structure in the soil
environment.
Real-Time PCR Assay for Detection and Enumeration ofDekkera bruxellensisin Wine
Authors: Phister Trevor G. , Mills David A.
Published: December 2003

Traditional methods to detect the spoilage yeastDekkera bruxellensisfrom wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerateD. bruxellensisin wine. Specific PCR primers toD. bruxellensiswere designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes ofD. bruxellensisinfection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage byD. bruxellensis.
Characterization of
Vibrio fluvialis
-LikeStrains Implicated in Limp LobsterDisease
Authors: Tall B. D. , Fall S. , Pereira M. R. , Ramos-Valle M. , Curtis S. K. , Kothary M. H. , Chu D. M. T. , Monday S. R. , Kornegay L. , Donkar T. , Prince D. , Thunberg R. L. , Shangraw K. A. , Hanes D. E. , Khambaty F. M. , Lampel K. A. , Bier J. W. , Bayer R. C.
Published: December 2003

Studies
were undertaken to characterize and determine the pathogenic mechanisms
involved in a newly described systemic disease in
Homarus
americanus
(American lobster) caused by a
Vibrio
fluvialis
-like microorganism. Nineteen isolates were obtained from
eight of nine lobsters sampled. Biochemically, the isolates resembled
V. fluvialis
, and the isolates grew optimally at 20°C;
none could grow at temperatures above 23°C. The type strain
(1AMA) displayed a thermal reduction time (D value) of 5.77 min at
37°C. All of the isolates required at least 1% NaCl for
growth. Collectively, the data suggest that these isolates may embody a
new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the
isolates revealed five closely related subgroups. Some isolates
produced a sheep hemagglutinin that was neither an outer membrane
protein nor a metalloprotease. Several isolates possessed
capsules. The isolates were highly susceptible to a variety of
antibiotics tested. However, six isolates were resistant to
erythromycin. Seventeen isolates harbored plasmids. Lobster challenge
studies revealed that the 50% lethal dose of a plasmid-positive
strain was 100-fold lower than that of a plasmid-negative strain,
suggesting that the plasmid may enhance the pathogenicity of these
microorganisms in lobsters. Microorganisms that were recovered from
experimentally infected lobsters exhibited biochemical and PFGE
profiles that were indistinguishable from those of the challenge
strain. Tissue affinity studies demonstrated that the challenge
microorganisms accumulated in heart and midgut tissues as well as in
the hemolymph. Culture supernatants and polymyxin B lysates of the
strains caused elongation of CHO cells in tissue culture, suggesting
the presence of a hitherto unknown enterotoxin. Both plasmid-positive
and plasmid-negative strains caused significant dose-related intestinal
fluid accumulations in suckling mice. Absence of viable organisms in
the intestinal contents of mice suggests that these microorganisms
cause diarrhea in mice by intoxication rather than by an infectious
process. Further, these results support the thermal reduction data at
37°C and suggest that the mechanism(s) that led to fluid
accumulation in mice differs from the disease process observed in
lobsters by requiring neither the persistence of viable microorganisms
nor the presence of plasmids. In summary, results of lobster studies
satisfy Koch's postulates at the organismal and molecular levels;
the findings support the hypothesis that these
V.
fluvialis
-like organisms were responsible for the originally
described systemic disease, which is now called limp lobster
disease.
BiologicalTraits ofXylella fastidiosaStrains from Grapes andAlmonds
Authors: Almeida Rodrigo P. P. , Purcell Alexander H.
Published: December 2003

Xylella fastidiosais a xylem-limited bacterium that causes various diseases, among them Pierce's disease of grapevine (PD) and almond leaf scorch (ALS). PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences amongX. fastidiosaisolated from these host plants. We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups ofX. fastidiosacaused ALS and overwintered within almonds after mechanical inoculation. Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD. However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains. Isolates able to cause both PD and ALS developed 10-fold-higher concentrations ofX. fastidiosain grapes than in almonds. In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes. We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines. Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media.
Identification and Population Dynamics of Yeasts inSourdough Fermentation Processes by PCR-Denaturing GradientGelElectrophoresis
Authors: Meroth Christiane B. , Hammes Walter P. , Hertel Christian
Published: December 2003

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence ofCandida humilis,Debaryomyces hansenii,Saccharomyces cerevisiae, andSaccharomyces uvarum. In sourdough A (traditional process with rye flour),C. humilisdominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively,S. cerevisiaebecame predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran,Candida kruseibecame dominant. Isolates identified asC. humilisandS. cerevisiaewere shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealedCladosporiumsp.,Saccharomyces servazii,S. uvarum, an unculturable ascomycete,Dekkera bruxellensis,Epicoccum nigrum, andS. cerevisiae.The last four species were also detected in sourdoughs A, B, and C.
Survival of
Helicobacter pylori
in a Natural Freshwater Environment
Authors: Adams B. L. , Bates T. C. , Oliver J. D.
Published: December 2003

The
mode by which
Helicobacter pylori
, the causative agent of most
gastric ulcers, is transmitted remains undetermined. Epidemiological
evidence suggests these organisms are waterborne; however,
H.
pylori
has rarely been grown from potential water sources. This
may be due to the ability of this organism to rapidly enter the viable
but nonculturable (VBNC) state. Our investigation examines the entrance
of
H. pylori
into this state in laboratory cultures and a
natural freshwater environment as well as the relationship between
morphology and culturability. To this end, membrane diffusion chambers
were utilized to expose the cells to the natural fluctuations of a
freshwater stream. In both the laboratory and environment, samples were
assayed for culturability using plate counts and stained using a
LIVE/DEAD
Bac
Light assay for viability and morphological
determinations. Additionally, water samples were collected, six
environmental parameters were measured, and resuscitation conditions
were examined.
H. pylori
was observed to lose culturability in
the laboratory and stream, although viability was maintained. While the
results of our study agree with those of previous studies which
suggested that there is a transition in morphology from rods to cocci
as culturability is lost, the morphological distribution of cells did
not change as culturability was lost in the environment. The majority
of cells in the VBNC state in the laboratory are cocci; however, all
morphological forms were present in the environment. The results of
these studies suggest that
H. pylori
persists in laboratory
cultures and the environment in the VBNC state and that cells in this
state represent a public health
hazard.
Enumeration and Characterization of Iron(III)-ReducingMicrobial Communities from Acidic Subsurface Sediments ContaminatedwithUranium(VI)
Authors: Petrie Lainie , North Nadia N. , Dollhopf Sherry L. , Balkwill David L. , Kostka Joel E.
Published: December 2003

Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml−1for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of theGeobacteraceaefamily, whereas a recently characterized Fe(III) reducer (Anaeromyxobactersp.) and organisms not previously shown to reduce Fe(III) (PaenibacillusandBrevibacillusspp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated thatGeobacteraceaesequences were more abundant in pristine compared to contaminated environments,whereasAnaeromyxobactersequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.
Single-Copy Green Fluorescent Protein Gene FusionsAllow Accurate Measurement of
Salmonella
Gene Expression InVitro and during Infection of MammalianCells
Authors: Hautefort Isabelle , Proença Maria José , Hinton Jay C. D.
Published: December 2003

We
developed a reliable and flexible green fluorescent protein (GFP)-based
system for measuring gene expression in individual bacterial cells.
Until now, most systems have relied upon plasmid-borne
gfp
gene fusions, risking problems associated with plasmid instability. We
show that a recently developed GFP variant, GFP
+
, is
suitable for assessing bacterial gene expression. Various

gfp
+

transcriptional fusions were
constructed and integrated as single copies into the chromosome of
Salmonella enterica
serovar Typhimurium. A comparison of the
expression levels of
proU-lacZ
and

proU-gfp
+

fusions showed that
GFP
+
reported
proU
activity in individual
Salmonella
cells as accurately as β-galactosidase
reported activity for entire populations. The single-copy

gfp
+

fusions were ideal for monitoring up-
and downregulation of
Salmonella
virulence genes. We
discovered that in vitro induction of the
SPI1
gene
prgH
occurs only in a portion of the population and that
the proportion varies with the growth phase. We determined the level of
expression of the SPI2 gene
ssaG
in bacteria released from
murine macrophages. Our results demonstrate for the first time that
single-copy GFP
+
fusions reliably report gene
expression in simple and complex environments. This approach promises
to allow accurate measurement of gene expression in individual bacteria
during animal
infection.
Role of the Glycine Betaine and Carnitine Transporters in Adaptation ofListeria monocytogenesto Chill Stress in Defined Medium
Authors: Angelidis Apostolos S. , Smith Gary M.
Published: December 2003

The food-borne pathogenListeria monocytogenesproliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation ofListeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate inL. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives ofL. monocytogenes10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter inL. monocytogenes10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.
Experimental Examination of BacteriophageLatent-Period Evolution as a Response to BacterialAvailability
Authors: Abedon Stephen T. , Hyman Paul , Thomas Cameron
Published: December 2003

For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (≥∼107bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is ∼70 to 80% of that of the wild type—which is nearly as short as the RB69 eclipse period—and which has a corresponding burst size that is ∼30% of that of the wild type. We show that at higher host densities (≥∼107bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (≤∼106bacteria/ml). From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished.