bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization of Community Structures and Sulfur Metabolism within Microbial Streamers in Japanese Hot Springs
Authors: Nakagawa Tatsunori , Fukui Manabu
Published: December 2003

Community
structures of submerged microbial slime streamers (SMSS) in
sulfide-containing hot springs at 72 to 80°C at Nakabusa and
Yumata, Japan, were investigated by molecular analysis based on the 16S
rRNA gene. The SMSS were classified into two consortia; consortium I
occurred at lower levels of sulfide in the hot springs (less than 0.1
mM), and consortium II dominated when the sulfide levels were higher
(more than 0.1 mM). The dominant cell morphotypes in consortium I were
filamentous and small rod-shaped cells. The filamentous cells
hybridized with fluorescent oligonucleotide probes for the domain
Bacteria
, the domain
Archaea
, and the family
Aquificaceae
. Our analysis of the denaturing gradient gel
electrophoresis (DGGE) bands by using reverse transcription (RT)-PCR
amplification with two primer sets (Eub341-F with the GC clamp and
Univ907R for the
Bacteria
and Eub341-F with the GC clamp and
Arch915R) indicated that dominant bands were phylogenetically related
to microbes in the genus
Aquifex
. On the other hand,
consortium II was dominated by long, small, rod-shaped cells, which
hybridized with the oligonucleotide probe S-*-Tdes-0830-a-A-20
developed in this study for the majority of as-yet-uncultivated
microbes in the class
Thermodesulfobacteria
. The dominant DGGE
band obtained by PCR and RT-PCR was affiliated with the genus
Sulfurihydrogenibium
. Moreover, our analysis of dissimilatory
sulfite reductase (DSR) gene sequences retrieved from both consortia
revealed a high frequency of DSR genes corresponding to the DSR of
Thermodesulfobacteria
-like microorganisms. Using both sulfide
monitoring and
35
SO
4
2−
tracer
experiments, we observed microbial sulfide production and consumption
by SMSS, suggesting that there is in situ sulfide production by
as-yet-uncultivated
Thermodesulfobacteria
-like microbes and
there is in situ sulfide consumption by
Sulfurihydrogenibium
-like microbes within the SMSS in the
Nakabusa and Yumata hot
springs.
Linked Redox Precipitation of Sulfur and Seleniumunder Anaerobic Conditions by Sulfate-Reducing BacterialBiofilms
Authors: Hockin Simon L. , Gadd Geoffrey M.
Published: December 2003

A
biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated
from a naturally occurring mixed biofilm and identified by 16S rDNA
analysis as a strain of
Desulfomicrobium norvegicum
, rapidly
removed 200 μM selenite from solution during growth on lactate
and sulfate. Elemental selenium and elemental sulfur were precipitated
outside SRB cells. Precipitation occurred by an abiotic reaction with
bacterially generated sulfide. This appears to be a generalized ability
among SRB, arising from dissimilatory sulfide biogenesis, and can take
place under low redox conditions and in the dark. The reaction
represents a new means for the deposition of elemental sulfur by SRB
under such conditions. A combination of transmission electron
microscopy, environmental scanning electron microscopy, and cryostage
field emission scanning electron microscopy were used to reveal the
hydrated nature of SRB biofilms and to investigate the location of
deposited sulfur-selenium in relation to biofilm elements. When
pregrown SRB biofilms were exposed to a selenite-containing medium,
nanometer-sized selenium-sulfur granules were precipitated within the
biofilm matrix. Selenite was therefore shown to pass through the
biofilm matrix before reacting with bacterially generated sulfide. This
constitutes an efficient method for the removal of toxic concentrations
of selenite from solution. Implications for environmental cycling and
the fate of sulfur and selenium are discussed, and a general model for
the potential action of SRB in selenium transformations is
presented.
High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus
Trichoderma reesei
Requires a Carrier Polypeptide with an Intact Domain Structure
Authors: Paloheimo Marja , Mäntylä Arja , Kallio Jarno , Suominen Pirkko
Published: December 2003

A
bacterial xylanase gene,
Nonomuraea flexuosa xyn11A
, was
expressed in the filamentous fungus
Trichoderma reesei
from
the strong cellobiohydrolase 1 promoter as fusions to a variety of
carrier polypeptides. By using single-copy isogenic transformants, it
was shown that production of this xylanase was clearly increased (up to
820 mg/liter) when it was produced as a fusion protein with a carrier
polypeptide having an intact domain structure compared to the
production (150 to 300 mg/liter) of fusions to the signal sequence
alone or to carriers having incomplete domain structures. The carriers
tested were the
T. reesei
mannanase I (Man5A, or
MANI) core-hinge and a fragment thereof and the cellulose
binding domain of
T. reesei
cellobiohydrolase II (Cel6A, or
CBHII) with and without the hinge region(s) and a fragment thereof. The
flexible hinge region was shown to have a positive effect on both the
production of Xyn11A and the efficiency of cleavage of the fusion
polypeptide. The recombinant Xyn11A produced had properties similar to
those of the native xylanase. It constituted 6 to 10% of the
total proteins secreted by the transformants. About three times more of
the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A
was observed. Even in the best Xyn11A producers, the levels of the
fusion mRNAs were only ∼10% of the level of
cel7A
(
cbh1
) mRNA in the untransformed host
strain.
Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus
Authors: González Tania , Terrón María C. , Zapico Ernesto J. , Téllez Alejandro , Yagüe Susana , Carbajo José M. , González Aldo E.
Published: December 2003

Laccases
produced by white rot fungi are involved in the degradation of lignin
and a broad diversity of other natural and synthetic molecules, having
a great potential for biotechnological applications. They are
frequently encoded by gene families, as in the basidiomycete
Trametes
sp. strain I-62, from which the
lcc1
,
lcc2
, and
lcc3
laccase genes have been cloned and
sequenced. A multiplex reverse transcription-PCR method to
simultaneously study the expression of these genes was developed in
this study. The assay proved to be quick, simple, highly sensitive, and
reproducible and is particularly valuable when numerous samples are to
be analyzed and/or if the amount of initial mRNA is limited. It was
used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl
alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and
3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in
Trametes
sp. strain I-62. These aromatic compounds produced
different induction patterns despite their chemical similarity. We
found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase
activity while also producing the highest increase in gene expression;
3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript
amounts of each gene fluctuated dramatically in the presence of these
three inducers, while the total amounts of laccase mRNAs seemed to be
modulated by a coordinated regulation of the different
genes.
Nitrification and Nitrifying Bacteria in the LowerSeine River and Estuary(France)
Authors: Cébron Aurélie , Berthe Thierry , Garnier Josette
Published: December 2003

The
Achères wastewater treatment plant, located just downstream of
Paris, discharges its effluents into the lower Seine River. The
effluents contain large numbers of heterotrophic bacteria, organic
matter, and ammonium and are a source of nitrifying bacteria. As a
result, degradation of organic matter by heterotrophic bacteria and
subsequent oxygen depletion occur immediately downstream of the
effluent outlet, whereas nitrifying bacteria apparently need to build
up a significant biomass before ammonium oxidation significantly
depletes the oxygen. We quantified the potential total nitrifying
activity and the potential activities of the ammonia- and
nitrite-oxidizing communities along the Seine River. In the summer, the
maximum nitrifying activity occurs in the upper freshwater estuary,∼
200 km downstream of Achères. The quantities of
nitrifying bacteria, based on
amoA
gene copy numbers, and of
Nitrobacter
organisms, based on 16S rRNA gene copy numbers,
were correlated with the potential nitrifying activities. The species
composition of ammonia-oxidizing bacteria was investigated at two
sites: the Triel station just downstream from Achères (km 84) and
the Seine freshwater estuary at the Duclair station (km 278). By means
of PCR primers targeting the
amoA
gene, a gene library was
created. Phylogenetic analysis revealed that the majority of the
analyzed clones at both sites were affiliated with the genus
Nitrosomonas
. The
Nitrosomonas oligotropha
- and
Nitrosomonas urea
-related clones represented nearly 81%
of the community of ammonia-oxidizing bacteria at Triel and 60%
at Duclair. Two other ammonia-oxidizing clusters of the β
subclass of the
Proteobacteria
, i.e.,
Nitrosomonas
europaea
- and
Nitrosospira
-like bacteria, were found in
smaller numbers. The major change in the ammonia-oxidizing community
between the two stations along the Seine River-upper estuary continuum
was the replacement of the
N. oligotropha
- and
N.
urea
-related bacteria by the
Nitrosospira
-affiliated
bacteria. Although the diversities of the ammonia oxidizers appear to
be similar for the two sites, only half of the restriction patterns are
common to both sites, which could be explained by the differences in
ammonium concentrations, which are much lower in the upper estuary than
in the river at the effluent outlet. These results imply a significant
immigration and/or selection of the ammonia-oxidizing bacterial
population along the continuum of the Seine River from Paris to the
estuary.
ControlledModulation of Folate Polyglutamyl Tail Length by Metabolic Engineeringof
Lactococcuslactis
Authors: Sybesma Wilbert , van den Born Erwin , Starrenburg Marjo , Mierau Igor , Kleerebezem Michiel , de Vos Willem M. , Hugenholtz Jeroen
Published: December 2003

The
dairy starter bacterium
Lactococcus lactis
is able to
synthesize folate and accumulates >90% of the produced
folate intracellularly, predominantly in the polyglutamyl form.
Approximately 10% of the produced folate is released into the
environment. Overexpression of
folC
in
L. lactis
led
to an increase in the length of the polyglutamyl tail from the
predominant 4, 5, and 6 glutamate residues in wild-type cells to a
maximum of 12 glutamate residues in the folate synthetase overproducer
and resulted in a complete retention of folate in the cells.
Overexpression of
folKE
, encoding the bifunctional protein
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and
GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl
tail length, leading to enhanced excretion of folate. By simultaneous
overexpression of
folKE
and
folC
, encoding the enzyme
folate synthetase or polyglutamyl folate synthetase, the average
polyglutamyl tail length was increased, again resulting in normal
wild-type distribution of folate. The production of bioavailable
monoglutamyl folate and almost complete release of folate from the
bacterium was achieved by expressing the gene for γ-glutamyl
hydrolase from human or rat origin. These engineering studies clearly
establish the role of the polyglutamyl tail length in intracellular
retention of the folate produced. Also, the potential application of
engineered food microbes producing folates with different tail lengths
is
discussed.
Efficient Degradation of 2,4,6-TrichlorophenolRequires a Set of Catabolic Genes Related to
tcp
Genesfrom
Ralstonia eutropha
JMP134(pJP4)
Authors: Matus V. , Sánchez M. A. , Martínez M. , González B.
Published: December 2003

2,4,6-Trichlorophenol
(2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are
known to degrade this compound. One of these,
Ralstonia
eutropha
JMP134(pJP4), a well-known, versatile chloroaromatic
compound degrader, is able to grow in 2,4,6-TCP by converting it to
2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate,
maleylacetate, and β-ketoadipate. Three enzyme activities
encoded by
tcp
genes, 2,4,6-TCP monooxygenase (
tcpA
),
6-chlorohydroxyquinol 1,2-dioxygenase (
tcpC
), and
maleylacetate reductase (
tcpD
), are involved in this catabolic
pathway. Here we provide evidence that all these
tcp
genes are
clustered in the
R. eutropha
JMP134(pJP4) chromosome, forming
the putative catabolic operon
tcpRXABCYD.
We studied the
presence of
tcp
-like gene sequences in several other
2,4,6-TCP-degrading bacterial strains and found two types of strains.
One type includes strains belonging to the
Ralstonia
genus and
possessing a set of
tcp
-like genes, which efficiently degrade
2,4,6-TCP and therefore grow in liquid cultures containing this
chlorophenol as a sole carbon source. The other type includes strains
belonging to the genera
Pseudomonas
,
Sphingomonas
, or
Sphingopixis
, which do not have
tcp
-like gene
sequences and degrade this pollutant less efficiently and which
therefore grow only as small colonies on plates with 2,4,6-TCP. Other
than strain JMP134, none of the bacterial strains whose genomes have
been sequenced possesses a full set of
tcp
-like gene
sequences.
Purification and Characterization ofα-l-Arabinopyranosidase andα-l-Arabinofuranosidase fromBifidobacteriumbreveK-110, a Human Intestinal AnaerobicBacterium Metabolizing Ginsenoside Rb2 andRc
Authors: Shin Ho-Young , Park Sun-Young , Sung Jong Hwan , Kim Dong-Hyun
Published: December 2003

Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizingBifidobacterium breveK-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+andp-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent onp-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent onp-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted onp-nitrophenyl-β-galactopyranoside orp-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobicBifidobacteriumsp.
Inactivation ofGeobacillus stearothermophilusSpores by High-Pressure CarbonDioxideTreatment
Authors: Watanabe Taisuke , Furukawa Soichi , Hirata Junichi , Koyama Tetsuya , Ogihara Hirokazu , Yamasaki Makari
Published: December 2003

High-pressure CO2treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores ofBacillus coagulans,Bacillus subtilis,Bacillus cereus,Bacillus licheniformis, andGeobacillus stearothermophiluswere subjected to CO2treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except theG. stearothermophilusspores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores ofG. stearothermophiluswere not the most resistant to CO2treatment. We also investigated the influence of temperature on CO2inactivation ofG. stearothermophilus.Treatment with CO2and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO2treatment ofG. stearothermophilusspores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2treatment ofG. stearothermophilusspores, CO2treatment at 95°C was more effective than treatment at 95°C alone.
Molecular Surveillance of Enterovirus andNorwalk-Like Virus in Oysters Relocated to a Municipal-Sewage-ImpactedGulfEstuary
Authors: Shieh Y. Carol , Baric Ralph S. , Woods Jacquelina W. , Calci Kevin R.
Published: December 2003

An
18-month survey was conducted to examine the prevalence of enteric
viruses and their relationship to indicators in environmentally
polluted shellfish. Groups of oysters, one group per 4 weeks, were
relocated to a coastal water area in the Gulf of Mexico that is
impacted by municipal sewage and were analyzed for enteroviruses,
Norwalk-like viruses (NLV), and indicator microorganisms (fecal
coliform,
Escherichia coli
, and male-specific coliphages). The
levels of indicator microorganisms were consistent with the expected
continuous pollution of the area. Fourteen of the 18 oyster samples
were found by reverse transcription (RT)-PCR to harbor NLV and/or
enterovirus sequences. Of the four virus-negative oysters, three had
exposure to water temperatures of >29°C. Concomitant
with these findings, two of these four oysters also accumulated the
lowest levels of coliphages. PCR primers targeting pan-enteroviruses
and the NLV 95/96-US common subset were utilized; NLV sequences were
detected more frequently than those of enteroviruses. Within the
12-month sampling period, NLV and enterovirus sequences were detected
in 58 and 42%, respectively, of the oysters (67% of the
oysters tested were positive for at least one virus) from a
prohibited shellfish-growing area approximately 30 m away
from a sewage discharge site. Eight (4.6%) of the 175 NLV capsid
nucleotide sequences were heterogeneous among the clones derived from
naturally polluted oysters. Overall, enteric viral sequences were found
in the contaminated oysters throughout all seasons except hot summer,
with a higher prevalence of NLV than enterovirus. Although a high
percentage of the oysters harbored enteric viruses, the virus levels
were usually less than or equal to 2 logs of RT-PCR-detectable units
per gram of oyster
meat.
Real-TimePCR Analysis of
Vibrio vulnificus
fromOysters
Authors: Campbell Mark S. , Wright Anita C.
Published: December 2003

Vibrio
vulnificus
is an opportunistic human pathogen commonly found in
estuarine environments. Infections are associated with raw oyster
consumption and can produce rapidly fatal septicemia in susceptible
individuals. Standard enumeration of this organism in shellfish or
seawater is laborious and inaccurate; therefore, more efficient assays
are needed. An oligonucleotide probe derived from the cytolysin gene,
vvhA
, was previously used for colony hybridizations to
enumerate
V. vulnificus
. However, this method requires
overnight growth, and vibrios may lack culturability under certain
conditions. In the present study, we targeted the same locus for
development of a TaqMan real-time PCR assay. Probe specificity was
confirmed by amplification of 28
V. vulnificus
templates and
by the lack of a PCR product with 22 non-
V. vulnificus
strains. Detection of
V. vulnificus
in pure cultures was
observed over a 6-log-unit linear range of concentration
(10
2
to 10
8
CFU ml
−1
), with a
lower limit of 72 fg of genomic DNA μl of PCR
mixture
−1
or the equivalent of six cells. Similar
sensitivity was observed in DNA extracted from mixtures of
V.
vulnificus
and
V. parahaemolyticus
cells. Real-time PCR
enumeration of artificially inoculated oyster homogenates correlated
well with colony hybridization counts (
r
2
=
0.97). Numbers of indigenous
V. vulnificus
cells in oysters by
real-time PCR showed no significant differences from numbers from plate
counts with probe (
t
test;
P
= 0.43). Viable
but nonculturable cells were also enumerated by real-time PCR and
confirmed by the
Bac
Light viability assay. These data indicate
that real-time PCR can provide sensitive species-specific detection and
enumeration of
V. vulnificus
in
seafood.
Identification and Characterization of BenzimidazoleResistance in
Monilinia fructicola
from Stone Fruit OrchardsinCalifornia
Authors: Ma Zhonghua , Yoshimura Michael A. , Michailides Themis J.
Published: December 2003

Low
and high levels of resistance to the benzimidazole fungicides benomyl
and thiophanate-methyl were observed in field isolates of
Monilinia
fructicola
, which is the causative agent of brown rot of stone
fruit. Isolates that had low levels of resistance (hereafter referred
to as LR isolates) and high levels of resistance (hereafter referred to
as HR isolates) were also cold and heat sensitive, respectively.
Results from microsatellite DNA fingerprints showed that genetic
identities among the populations of sensitive (S), LR, and HR isolates
were very high (>0.96). Analysis of DNA sequences of theβ
-tubulin gene showed that the LR isolates had a point mutation
at codon 6, causing a replacement of the amino acid histidine by
tyrosine. Codon 198, which encodes a glutamic acid in S and LR
isolates, was converted to a codon for alanine in HR isolates. Based on
these point mutations in the β-tubulin gene,
allele-specific PCR assays were developed for rapid detection of
benzimidazole-resistant isolates of
M.
fructicola
from stone
fruit.
Prevalence and Antimicrobial Resistance of
Enterococcus
Species Isolated from RetailMeats
Authors: Hayes Joshua R. , English Linda L. , Carter Peggy J. , Proescholdt Terry , Lee Kyung Y. , Wagner David D. , White David G.
Published: December 2003

From
March 2001 to June 2002, a total of 981 samples of retail raw meats
(chicken, turkey, pork, and beef) were randomly obtained from 263
grocery stores in Iowa and cultured for the presence of
Enterococcus
spp. A total of 1,357 enterococcal isolates were
recovered from the samples, with contamination rates ranging from
97% of pork samples to 100% of ground beef samples.
Enterococcus faecium
was the predominant species recovered
(61%), followed by
E. faecalis
(29%), and
E.
hirae
(5.7%).
E. faecium
was the predominant
species recovered from ground turkey (60%), ground beef
(65%), and chicken breast (79%), while
E.
faecalis
was the predominant species recovered from pork chops
(54%). The incidence of resistance to many production and
therapeutic antimicrobials differed among enterococci recovered from
retail meat samples. Resistance to quinupristin-dalfopristin, a human
analogue of the production drug virginiamycin, was observed in 54, 27,
9, and 18% of
E. faecium
isolates from turkey, chicken,
pork, and beef samples, respectively. No resistance to linezolid or
vancomycin was observed, but high-level gentamicin resistance was
observed in 4% of enterococci, the majority of which were
recovered from poultry retail meats. Results indicate that
Enterococcus
spp. commonly contaminate retail meats and that
dissimilarities in antimicrobial resistance patterns among enterococci
recovered from different meat types may reflect the use of approved
antimicrobial agents in each food animal production
class.
Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by
Pseudomonas fluorescens
Authors: de Souza Jorge T. , de Boer Marjan , de Waard Pieter , van Beek Teris A. , Raaijmakers Jos M.
Published: December 2003

Zoospores
play an important role in the infection of plant and animal hosts by
oomycetes and other zoosporic fungi. In this study, six fluorescent
Pseudomonas
isolates with zoosporicidal activities were
obtained from the wheat rhizosphere. Zoospores of multiple oomycetes,
including
Pythium
species,
Albugo candida
, and
Phytophthora infestans
, were rendered immotile within
30 s of exposure to cell suspensions or cell culture
supernatants of the six isolates, and subsequent lysis occurred within
60 s. The representative strain SS101, identified as
Pseudomonas fluorescens
biovar II, reduced the surface tension
of water from 73 to 30 mN m
−1
. The application of
cell suspensions of strain SS101 to soil or hyacinth bulbs provided
significant protection against root rot caused by
Pythium
intermedium
. Five Tn
5
mutants of strain SS101lacked the abilities to reduce the surface tension of water and to
cause lysis of zoospores. Genetic characterization of two
surfactant-deficient mutants showed that the transposons had integrated
into condensation domains of peptide synthetases. A partially purified
extract from strain SS101 reduced the surface tension of water to 30 mN
m
−1
and reached the critical micelle concentration
at 25 μg ml
−1
. Reverse-phase
high-performance liquid chromatography yielded eight different
fractions, five of which had surface activity and caused lysis of
zoospores. Mass spectrometry and nuclear magnetic resonance analyses
allowed the identification of the main constituent as a cyclic
lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon
hydroxy fatty acid. The other four zoosporicidal fractions were closely
related to the main constituent, with molecular massesranging from 1,111 to 1,169
Da.
Heat Shock Treatment Increases the Frequency of Lossof an Erythromycin Resistance-Encoding Transposable Element from theChromosome of
Lactobacillus crispatus
CHCC3692
Authors: Strøman Per , Müller Christina C. , Sørensen Kim I.
Published: December 2003

A
3,165-bp chromosomally integrated transposon, designatedTn
3692
, of the gram-positive strain
Lactobacillus
crispatus
CHCC3692 contains an
erm
(B) gene
conferring resistance to erythromycin at concentrations of up to 250μ
g/ml. Loss of this resistance can occur spontaneously, but the
rate is substantially increased by heat shock treatment. Heat shock
treatment at 60°C resulted in an almost 40-fold increase in the
frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047μ
g/ml). The phenotypic change was followed by a dramatic
increase in transcription of the transposase gene and the concomitant
loss of an approximately 2-kb DNA fragment carrying the
erm
(B)
gene from the 3,165-bp
erm
transposon. In cells that were not
subjected to heat shock, transcription of the transposase gene was not
detectable. The upstream sequence of the transposase gene did not show
any homology to known heat shock promoters in the gene data bank.
Significant homology (>99%) was observed between the
erythromycin resistance-encoding gene from
L. crispatus
CHCC3692 and the
erm
(B) genes from other gram-positive
bacteria, such as
Streptococcus agalactiae
,
Streptococcus
pyogenes
,
Enterococcus faecium
, and
Lactobacillus
reuteri
, which strongly indicates a common origin of the
erm
(B) gene for these species. The transposed DNA element was
not translocated to other parts of the genome of CHCC3692, as
determining by Southern blotting, PCR analysis, and DNA sequencing. No
other major aberrations were observed, as judged by colony morphology,
growth performance of the strain, and pulsed-field gel electrophoresis.
These observations suggest that heat shock treatment could be used as a
tool for the removal of unwanted antibiotic resistance genes harbored
in transposons flanked by insertion sequence elements or transposases
in lactic acid bacteria used for animal and human food
production.
Assessment of an Enterovirus Sewage SurveillanceSystem by Comparison of Clinical Isolates with Sewage Isolatesfrom Milwaukee, Wisconsin, Collected August 1994 toDecember2002
Authors: Sedmak Gerald , Bina David , MacDonald Jeffrey
Published: December 2003

The
quantity and serotypes of enteroviruses (EVs) in the influent of a
local sewage treatment plant were compared to local clinical EV cases
to determine if testing of sewage is adequate for an EV surveillance
system. The study was carried out from August 1994 to December 2002.
Monthly influent specimens were processed by organic flocculation, and
dilutions of concentrate were inoculated onto a number of different
cell types for virus isolation. EVs were detected in 88 of 100 monthly
influent samples. Sewage EV titers were calculated by using software
provided by the U.S. Environmental Protection Agency for
most-probable-number determination. All 1,068 sewage EV isolates were
further grouped (echovirus, coxsackievirus B, coxsackievirus A, or
poliovirus) by cell culture host range analysis (growth pattern of
isolates on passage to seven cell lines), and 39.0% of the 1,022
EV isolates categorized as non-poliovirus EVs were specifically
serotyped. For clinical cases, primary virus isolation tests were
performed on specimens submitted by local hospitals and EV isolates
submitted by hospitals were serotyped. Clinical EVs were documented for
81 of the 100 months studied. In all, 694 EV isolates from clinical
cases were serotyped. Annually, between 4 and 11 different serotypes of
non-poliovirus EVs were identified in sewage and from 9 to 19 different
non-poliovirus EV serotypes were identified from clinical specimens.
Usually, the most commonly detected sewage EV serotypes were similar to
the most commonly detected clinical serotypes; e.g., for 1997,
echovirus 6 accounted for 53.1% of the typed sewage isolates and
39.4% of the clinical infections, while in 1998, echovirus 30
accounted for 50.0 and 46.1%, respectively. In 1999,
60.3% of the EVs from clinical cases and 79.7% of the
sewage isolates were echovirus 11; in 2000, 33.3% of the EVs
from clinical cases and 40.7% of the sewage isolates were
coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical
cases and 36.2% of the sewage isolates were echovirus 13. Annual
peaks of both sewage EV titers and clinical cases occurred in late
summer or early fall. In some years, early spring sewage EVs
portended some of the EVs that would predominate clinically
during the following
summer.
Inoculum Size Influences Bacterial CrossContamination betweenSurfaces
Authors: Montville R. , Schaffner D. W.
Published: December 2003

Many
factors have been shown to influence bacterial transfer between
surfaces, including surface type, bacterial species, moisture level,
pressure, and friction, but the effect of inoculum size on bacterial
transfer has not yet been established. Bacterial cross contamination
rates during performance of common food service tasks were previously
determined in our laboratory using nalidixic acid-resistant
Enterobacter aerogenes
. Eight different transfer rates were
determined, each involving a minimum of 30 volunteers. The influence of
source inoculum level on the percentage of bacteria transferred
(percent transfer rates) and log
10
CFU per recipient surface
was determined using statistical analysis. The effect of inoculum size
on transfer rate was highly statistically significant (
P
< 0.0001) for all transfer rate data combined (352
observations) and for each individual cross contamination rate, except
for data on contamination via transfer from chicken to hand through a
glove barrier (
P
= 0.1643). Where inoculum size on the
source was greater, transfer rates were lower, and where inoculum size
on the source was less, transfer rates were higher. The negative linear
trend was more obvious for activities that had a larger range of
inoculum sizes on the source surface. This phenomenon has serious
implications for research seeking to determine bacterial cross
contamination rates, since the different transfer efficiencies that
were previously shown to be associated with certain activities may
actually be the result of differing initial inoculum levels. The
initial inoculum size on the source and the amount of bacteria
transferred must both be considered to accurately determine bacterial
transfer
rates.
Functional Characterization of a Composite BacteriocinLocus from Malt Isolate
Lactobacillus sakei
5
Authors: Vaughan Anne , Eijsink Vincent G. H. , van Sinderen Douwe
Published: December 2003

Lactobacillus
sakei
5, isolated from malted barley, produces three bacteriocins.
Genetic and functional analysis of the purified bacteriocins showed
that this strain produces a plasmid-encoded bacteriocin that is
identical to sakacin P, as well as two novel, chromosomally encoded
bacteriocins, which were designated sakacin T and sakacin X. The
structural genes specifying sakacin T and sakacin X are part of the
sakacin TX
locus, which consists of two adjacent but
divergently oriented gene clusters. The first gene cluster includes
stxP
,
stxR
,
stxK
, and
stxT
, which,
based on functional and comparative sequence analysis, are believed to
encode an inducing peptide and proteins involved in regulation and
secretion of these bacteriocins. The second gene cluster includes the
structural and immunity genes for sakacin T, a class IIb two-peptide
bacteriocin composed of SakT
α
and
SakT
β
, and sakacin X, a class IIa bacteriocin.
Interestingly, a so-called transport accessory protein was absent from
the locus, and based on our results it appears that a dedicated
accessory protein is not required for processing and transport of
sakacin T and sakacin
X.
BacterialSynergism or Antagonism in a Gel CassetteSystem
Authors: Tsigarida Eirini , Boziaris Ioannis S. , Nychas George-John E.
Published: December 2003

The
growth and the metabolic activity of
Shewanella putrfaciens
,
Brochothrix thermosphacta
, and
Pseudomonas
sp., when
cultured individually or in all possible combinations in gel cassettes
system supplemented with 0.1% glucose at 5°C, were
investigated. The overall outcome was that the coexistence of the
above-mentioned microorganisms affected not only each growth rate but
also their type of metabolic end products compared to the control
cultures. These effects were varied and depended on the selection of
the combination of the tested bacteria. For example, the growth of
Pseudomonas
sp. strains cocultured with either
B.
thermosphacta
or
S. putrefaciens
strains resulted in
different effects: a promoting one for the first and an inhibitory one
for the second. Moreover, the production of formic acid and two
unidentified organic acids (peaks a and b) was characteristic in all
cases in which
S. putrefaciens
was
cultured.
Laboratory Cultivation of Widespread and PreviouslyUncultured SoilBacteria
Authors: Joseph Shayne J. , Hugenholtz Philip , Sangwan Parveen , Osborne Catherine A. , Janssen Peter H.
Published: December 2003

Most
soil bacteria belong to family-level phylogenetic groups with few or no
known cultivated representatives. We cultured a collection of 350
isolates from soil by using simple solid media in petri dishes. These
isolates were assigned to 60 family-level groupings in nine bacterial
phyla on the basis of a comparative analysis of their 16S rRNA genes.
Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed
family-level groupings, many from poorly studied bacterial classes and
phyla. They included members of subdivisions 1, 2, 3, and 4 of the
phylum
Acidobacteria
, subdivision 3 of the phylum
Verrucomicrobia
, subdivision 1 of the phylum
Gemmatimonadetes
, and subclasses
Acidimicrobidae
and
Rubrobacteridae
of the phylum
Actinobacteria
. In
addition, members of 10 new family-level groupings of subclass
Actinobacteridae
of the phylum
Actinobacteria
and
classes
Alphaproteobacteria
,
Betaproteobacteria
, and
Gammaproteobacteria
of the phylum
Proteobacteria
were
obtained. The high degree of phylogenetic novelty and the number of
isolates affiliated with so-called unculturable groups show that simple
cultivation methods can still be developed further to obtain laboratory
cultures of many phylogenetically novel soil
bacteria.
Microbial Communities Associated with GeologicalHorizons in Coastal Subseafloor Sediments from the Sea ofOkhotsk
Authors: Inagaki Fumio , Suzuki Masae , Takai Ken , Oida Hanako , Sakamoto Tatsuhiko , Aoki Kaori , Nealson Kenneth H. , Horikoshi Koki
Published: December 2003

Microbial
communities from a subseafloor sediment core from the southwestern Sea
of Okhotsk were evaluated by performing both cultivation-dependent and
cultivation-independent (molecular) analyses. The core, which extended
58.1 m below the seafloor, was composed of pelagic clays with
several volcanic ash layers containing fine pumice grains. Direct cell
counting and quantitative PCR analysis of archaeal and bacterial 16S
rRNA gene fragments indicated that the bacterial populations in the ash
layers were approximately 2 to 10 times larger than those in the clays.
Partial sequences of 1,210 rRNA gene clones revealed that there were
qualitative differences in the microbial communities from the two
different types of layers. Two phylogenetically distinct archaeal
assemblages in the
Crenarchaeota
, the miscellaneous
crenarchaeotic group and the deep-sea archaeal group, were the most
predominant archaeal 16S rRNA gene components in the ash layers and the
pelagic clays, respectively. Clones of 16S rRNA gene sequences from
members of the gamma subclass of the class
Proteobacteria
dominated the ash layers, whereas sequences from members of the
candidate division OP9 and the green nonsulfur bacteria dominated the
pelagic clay environments. Molecular (16S rRNA gene sequence) analysis
of 181 isolated colonies revealed that there was regional proliferation
of viable heterotrophic mesophiles in the volcanic ash layers, along
with some gram-positive bacteria and actinobacteria. The porous ash
layers, which ranged in age from tens of thousands of years to hundreds
of thousands of years, thus appear to be discrete microbial habitats
within the coastal subseafloor clay sediment, which are capable of
harboring microbial communities that are very distinct from the
communities in the more abundant pelagic
clays.
Targeting Methanopterin Biosynthesis ToInhibitMethanogenesis
Authors: Dumitru Razvan , Palencia Hector , Schroeder Scott D. , DeMontigny Bree A. , Takacs James M. , Rasche Madeline E. , Miner Jess L. , Ragsdale Stephen W.
Published: December 2003

This
paper describes the design, synthesis, and successful employment of
inhibitors of
4-(β-
d
-ribofuranosyl)aminobenzene-5′-phosphate
(RFA-P) synthase, which catalyzes the first committed step in the
biosynthesis of methanopterin, to specifically halt the growth of
methane-producing microbes. RFA-P synthase catalyzes the first step in
the synthesis of tetrahydromethanopterin, a key cofactor required for
methane formation and for one-carbon transformations in methanogens. A
number of inhibitors, which are N-substituted derivatives of
p
-aminobenzoic acid (pABA), have been synthesized and their
inhibition constants with RFA-P synthase have been determined. Based on
comparisons of the inhibition constants among various inhibitors, we
propose that the pABA binding site in RFA-P synthase has a relatively
large hydrophobic pocket near the amino group. These enzyme-targeted
inhibitors arrest the methanogenesis and growth of pure cultures of
methanogens. Supplying pABA to the culture relieves the inhibition,
indicating a competitive interaction between pABA and the inhibitor at
the cellular target, which is most likely RFAP synthase. The inhibitors
do not adversely affect the growth of pure cultures of the bacteria
(acetogens) that play a beneficial role in the rumen. Inhibitors added
to dense ruminal fluid cultures (artificial rumena) halt
methanogenesis; however, they do not inhibit volatile fatty acid (VFA)
production and, in some cases, VFA levels are slightly elevated in the
methanogenesis-inhibited cultures. We suggest that inhibiting
methanopterin biosynthesis could be considered in strategies to
decrease anthropogenic methane emissions, which could have an
environmental benefit since methane is a potent greenhouse
gas.
Transduction of Porcine Enteropathogenic
Escherichia coli
with a Derivative of a Shiga Toxin 2-EncodingBacteriophage in a Porcine Ligated Ileal LoopSystem
Authors: Tóth István , Schmidt Herbert , Dow Mohamed , Malik Anna , Oswald Eric , Nagy Béla
Published: December 2003

In
this study, we have investigated the ability of detoxified Shiga toxin
(Stx)-converting bacteriophages Φ3538
(Δ
stx
2
::
cat
)
(H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861,
1999) and H-19B::Tn
10d-bla
(D.
W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to
lysogenize enteropathogenic
Escherichia coli
(EPEC) strains in
vivo. We were able to transduce the porcine EPEC strain 1390 (O45) withΦ
3538
(Δ
stx
2
::
cat
)
in porcine ligated ileal loops but not the human EPEC prototype strain
E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was
lysogenized under these in vivo conditions when
E. coli
K-12
containing H-19B::Tn
10d-bla
was used as
the
stx1
phage donor. The repeated success in the in vivo
transduction of an Stx2-encoding phage to a porcine EPEC strain in pig
loops was in contrast to failures in the in vitro trials with these and
other EPEC strains. These results indicate that in vivo conditions are
more effective for transduction of Stx2-encoding phages than in vitro
conditions.
Molecular andCulture-Based Analyses of Aerobic Carbon Monoxide OxidizerDiversity†
Authors: King Gary M.
Published: December 2003

Isolates
belonging to six genera not previously known to oxidize CO were
obtained from enrichments with aquatic and terrestrial plants. DNA from
these and other isolates was used in PCR assays of the gene for the
large subunit of carbon monoxide dehydrogenase (
coxL
).
CoxL
and putative
coxL
fragments were amplified from
known CO oxidizers (e.g.,
Oligotropha carboxidovorans
and
Bradyrhizobium japonicum
), from novel CO-oxidizing isolates
(e.g.,
Aminobacter
sp. strain COX,
Burkholderia
sp.
strain LUP,
Mesorhizobium
sp. strain NMB1,
Stappia
strains M4 and M8,
Stenotrophomonas
sp. strain LUP, and
Xanthobacter
sp. strain COX), and from several well-known
isolates for which the capacity to oxidize CO is reported here for the
first time (e.g.,
Burkholderia fungorum
LB400,
Mesorhizobium loti
,
Stappia stellulata
, and
Stappia aggregata
). PCR products from several taxa, e.g.,
O. carboxidovorans
,
B. japonicum
, and
B.
fungorum
, yielded sequences with a high degree
(>99.6%) of identity to those in GenBank or genome
databases. Aligned sequences formed two phylogenetically distinct
groups. Group OMP contained sequences from previously known
CO oxidizers, including
O. carboxidovorans
and
Pseudomonas
thermocarboxydovorans
, plus a number of closely
related sequences. Group BMS was dominated by putative
coxL
sequences from genera in the
Rhizobiaceae
and otherα
-
Proteobacteria
. PCR analyses revealed that many CO
oxidizers contained two
coxL
sequences, one from each group.
CO oxidation by
M. loti
, for which whole-genome
sequencing has revealed a single BMS-group putative
coxL
gene,
strongly supports the notion that BMS sequences represent functional CO
dehydrogenase proteins that are related to but distinct from previously
characterized aerobic CO
dehydrogenases.
Uptake ofCarbon Monoxide and Hydrogen at Environmentally Relevant Concentrationsby Mycobacteria†
Authors: King Gary M.
Published: December 2003

Liquid
culture assays revealed a previously unreported capacity
for
Mycobacterium bovis
BCG,
M. gordonae
,
and
M. marinum
to oxidize CO and for
M
.
smegmatis
to consume molecular hydrogen.
M
.
bovis
BCG,
M. gordonae
,
M
.
smegmatis
, and
M. tuberculosis
H37Ra oxidized CO at
environmentally relevant concentrations (<50 ppm);
H
2
oxidation by
M. gordonae
and
M. smegmatis
also occurred at environmentally
relevant concentrations (<10 ppm). CO was not consumed by
M. avium
or
M. microti
, although
the latter appeared to possess CO dehydrogenase (CODH) genes based on
PCR results with primers designed for the CODH large subunit,
coxL. M. smegmatis
and
M
.
gordonae
oxidized CO under suboxic (10 and 1%
atmospheric oxygen) and anoxic conditions in the presence of nitrate;
no oxidation occurred under anoxic conditions without nitrate. Similar
results were obtained for H
2
oxidation by
M
.
smegmatis
. Phylogenetic analyses of
coxL
PCR products
indicated that mycobacterial sequences form a subclade distinct from
that of other bacterial
coxL
, with limited differentiation
among fast- and slow-growing
strains.
Characterization of the
argA
Gene Requiredfor Arginine Biosynthesis and Syringomycin Production by
Pseudomonas syringae
pv.syringae
Authors: Lu Shi-En , Soule Jonathan D. , Gross Dennis C.
Published: December 2003

Two
types of necrosis-inducing lipodepsipeptide toxins, called syringomycin
and syringopeptin, are major virulence factors of
Pseudomonas
syringae
pv. syringae strain B301D. A previous study showed that a
locus, called
syrA
, was required for both syringomycin
production and plant pathogenicity, and the
syrA
locus was
speculated to encode a regulator of toxin production. In this study,
sequence analysis of the 8-kb genomic DNA fragment that complements the
syrA
phenotype revealed high conservation among a broad
spectrum of fluorescent pseudomonads. The putative protein encoded by
open reading frame 4 (ORF4) (1,299 bp) in the
syrA
locus
region exhibited 85% identity to ArgA, which is involved in
arginine biosynthesis in
Pseudomonas aeruginosa
. Growth of
strain W4S2545, the
syrA
mutant, required supplementation of N
minimal medium with arginine. Similarly, syringomycin production of
syrA
mutant W4S2545 was restored by the addition of arginine
to culture media. Furthermore, the insertion of Tn
5
in the
genome of the
syrA
mutant W4S2545 was localized between
nucleotides 146 and 147 in ORF4, and syringomycin production was
complemented in
trans
with the wild-type DNA fragment
containing intact ORF4. These results demonstrate that the
syrA
locus is the
argA
gene of
P. syringae
pv. syringae and that
argA
is directly involved in arginine
biosynthesis and therefore indirectly affects syringomycin production
because of arginine
deficiency.
Abilityof
Lactococcus lactis
To Export Viral CapsidAntigens: a Crucial Step for Development of LiveVaccines
Authors: Dieye Yakhya , Hoekman Arjan J. W. , Clier Florence , Juillard Vincent , Boot Hein J. , Piard Jean-Christophe
Published: December 2003

Thefood grade bacterium
Lactococcus lactis
is a
potential vehicle for protein delivery in the gastrointestinal tract.
As a model, we constructed lactococcal strains producing antigens of
infectious bursal disease virus (IBDV). IBDV infects chickens and
causes depletion of B-lymphoid cells in the bursa of Fabricius and
subsequent immunosuppression, morbidity, or acute mortality. The two
major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were
expressed and targeted to the cytoplasm, the cell wall, or the
extracellular compartment of
L. lactis
. Whereas VP3
was successfully targeted to the three compartments by the use of
relevant expression and export vectors, VP2 was recalcitrant to export,
thus confirming the difficulty of translocating naturally nonsecreted
proteins across the bacterial membrane. This defect could be partly
overcome by fusing VP2 to a naturally secreted protein (the
staphylococcal nuclease Nuc) that carried VP2 through the membrane.
Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial
compartments were administered orally to chickens. The chickens did not
develop any detectable immune response against VP2 and VP3 but did
exhibit an immune response against Nuc when Nuc-VP2 was anchored to the
cell wall of
lactococci.