bluuebunny/crossref_metadata_2025 huggingface dataset:
Activin A induces craniofacial cartilage from undifferentiated
Xenopus
ectoderm
in
vitro
Authors: Furue Miho , Myoishi Yasufumi , Fukui Yasuto , Ariizumi Takashi , Okamoto Tetsuji , Asashima Makoto
Published: November 2002

Activin A has potent mesoderm-inducing activity in amphibian embryos and induces various mesodermal tissues
in vitro
from the isolated presumptive ectoderm. By using a sandwich culture method established to examine activin A activity, we previously demonstrated that activin-treated ectoderm can function as both a head and trunk-tail organizer, depending on the concentration of activin A. By using activin A and undifferentiated presumptive ectoderm, it is theoretically possible to reproduce embryonic induction. Here, we test this hypothesis by studying the induction of cartilage tissue by using the sandwich-culture method. In the sandwiched explants, the mesenchymal cell condensation expressed type II collagen and cartilage homeoprotein-1 mRNA, and subsequently, cartilage was induced as they are
in vivo. goosecoid
(
gsc
) mRNA was prominently expressed in the cartilage in the explants.
Xenopus distal-less 4
(
X-dll4
) mRNA was expressed throughout the explants. In
Xenopus
embryos,
gsc
expression is restricted to the cartilage of the lower jaw, and
X-dll4
is widely expressed in the ventral head region, including craniofacial cartilage. These finding suggest that the craniofacial cartilage, especially lower jaw cartilage, was induced in the activin–treated sandwiched explants. In addition, a normal developmental pattern was recapitulated at the histological and genetic level. This work also suggests that the craniofacial cartilage-induction pathway is downstream of activin A. This study presents a model system suitable for the
in vitro
analysis of craniofacial cartilage induction in vertebrates.
Optical imaging reveals retinotopic organization of dorsal V3 in New World owl monkeys
Authors: Lyon David C. , Xu Xiangmin , Casagrande Vivien A. , Stefansic James D. , Shima Daniel , Kaas Jon H.
Published: November 2002

Optical imaging of intrinsic responses to visual stimuli in extrastriate cortex of owl monkeys provided evidence for the dorsal half of the third visual area, V3. Visual stimuli were used to selectively activate locations in dorsolateral V2 and the rostrally adjoining presumptive V3. Consistent with the proposed retinotopies of dorsal V2 and dorsal V3, small bars of drifting gratings along the horizontal meridian of the contralateral hemifield activated cortex along the V2/V3 border, whereas such stimuli along the vertical meridian activated cortex along the rostral border of V3. Stimuli in limited locations in the lower visual quadrant revealed mirror reversals of retinotopy in dorsal V2 and V3, whereas stimuli in the upper visual quadrant failed to activate either region. Brain sections processed for cytochrome oxidase from the same cases provided architectonic borders of V2 that matched those indicated by the optical imaging. The results support the concept that a narrow dorsal V3 exists in monkeys. V3d borders dorsal V2 and contains a smaller, mirror-image representation of the lower visual quadrant.
Host–pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of
Salmonella
pathogenicity island-2 virulence genes
Authors: Zaharik Michelle L. , Vallance Bruce A. , Puente José L. , Gros Philippe , Finlay B. Brett
Published: November 2002

Nramp1
(
N
atural
r
esistance-
a
ssociated
m
acrophage
p
rotein-
1
; also known as Slc11a1) is a host resistance gene that provides protection against several intracellular pathogens, including
Salmonella
enterica
serovar Typhimurium. Little is known about the dynamic interplay that occurs between mammalian host resistance determinants such as Nramp1 and pathogens during infection. To explore these interactions, we examined the effect of Nramp1 on expression of
Salmonella typhimurium
(STM) virulence factors. We demonstrate that
Salmonella
pathogenicity island
2
(SPI2) is essential for replication of STM in spleens of infected
Nramp1

+/+

mice. Furthermore, the presence of Nramp1 in transfected cell lines and congenic knockout mice resulted in the up-regulation of STM SPI2-associated virulence genes critical for intramacrophage survival. This Nramp1-dependent up-regulation of SPI2 was mimicked
in vitro
by chelation of iron, demonstrating the iron-responsive nature of expression of STM SPI2-associated virulence genes. We propose that acquisition of SPI2 by
S. enterica
not only enabled this bacterium to become an effective intracellular pathogen but also allowed the bacterium to withstand the effects of macrophage defense mechanisms such as Nramp1 early in the evolution of its pathogenic character. These dynamic Nramp1–pathogen interactions may be essential for regulating the course of an infection. This study demonstrates the presence of a previously undescribed direct influence of a mammalian innate host resistance locus on a pathogen at the genetic level.
Cultivating the uncultured
Authors: Zengler Karsten , Toledo Gerardo , Rappé Michael , Elkins James , Mathur Eric J. , Short Jay M. , Keller Martin
Published: November 2002

The recent application of molecular phylogeny to environmental samples has resulted in the discovery of an abundance of unique and previously unrecognized microorganisms. The vast majority of this microbial diversity has proved refractory to cultivation. Here, we describe a universal method that provides access to this immense reservoir of untapped microbial diversity. This technique combines encapsulation of cells in gel microdroplets for massively parallel microbial cultivation under low nutrient flux conditions, followed by flow cytometry to detect microdroplets containing microcolonies. The ability to grow and study previously uncultured organisms in pure culture will enhance our understanding of microbial physiology and metabolic adaptation and will provide new sources of microbial metabolites. We show that this technology can be applied to samples from several different environments, including seawater and soil.
Dynamic behaviour of the keyhole in laser welding
Authors: J Kroos  , U Gratzke  , M Vicanek  , G Simon
Published: November 2002

The dynamic behaviour of a keyhole in laser welding is studied theoretically. Starting from the stationary state, where the recoil pressure from ablating particles is in equilibrium with the surface tension at the keyhole wall, the collapse time due to a sudden laser shut-down is calculated. The characteristic time constant (r03rho / gamma )1/2of the system (r0is the initial keyhole radius, rho is the density of the melt, gamma is the gamma coefficient of surface tension) which is approximately 0.1 ms for Al, Fe and Cu turns out to be a lower limit of the keyhole closing time. Linear stability analysis of the stationary state reveals that under conditions relevant in practice, the keyhole is expected to perform oscillations with frequencies of several hundred Hertz. The results of this investigation are particularly important for pulsed laser applications.
A Multiresistant Clone of Shiga Toxin-Producing
Escherichia coli
O118:[H16] Is Spread in Cattle and Humans over Different European Countries
Authors: Maidhof Heinrich , Guerra Beatriz , Abbas Sascha , Elsheikha Hany M. , Whittam Thomas S. , Beutin Lothar
Published: November 2002

Multiresistant Shiga toxin-producing
Escherichia coli
(STEC) O118:H16 and O118 nonmotile strains (designated O118:[H16]) were detected by examination of 171 STEC isolates for their antimicrobial sensitivity. Of 48 STEC O118:[H16] strains, 98% were resistant to sulfonamide, 96% were resistant to streptomycin, 79% were resistant to kanamycin, 75% were resistant to tetracycline, 67% were resistant to ampicillin, 60% were resistant to chloramphenicol, 48% were resistant to trimethoprim, and 10% each were resistant to gentamicin and nalidixic acid. Nalidixic acid resistance and reduced susceptibility to ciprofloxacin were associated with the mutation
gyrA
LEU-83
. The STEC O118:[H16] strains were found to belong to a single genetic clone as investigated by multilocus enzyme electrophoresis and by multilocus sequence analysis of
E. coli
housekeeping genes. The STEC O118:[H16] strains originated from humans and cattle and were isolated in seven different countries of Europe between 1986 and 1999. Strains showing multiresistance to up to eight different antimicrobials predominated among the more recent STEC O118:[H16] strains. The genes in parentheses were associated with resistance to kanamycin (
aphA1-Ia
), chloramphenicol (
catA1
), tetracycline [
tet
(A)], and ampicillin (
bla
TEM-1
). Class 1 integrons containing
sulI
(sulfonamide resistance),
aadA1a
(streptomycin resistance), or
dfrA1
(trimethoprim resistance)
-aadA1a
gene cassettes were detected in 28 strains. The
bla
TEM-1b
gene was present in 18 of 21 strains that were examined by nucleotide sequencing. Class 1 integrons and
bla
TEM
genes were localized on plasmids and/or on the chromosome in different STEC O118:[H16] strains. Hybridization of
Xba
I-digested chromosomal DNA separated by pulsed-field gel electrophoresis revealed that
bla
TEM
genes were integrated at different positions in the chromosome of STEC O118:[H16] strains that could have occurred by Tn
2
insertion. Our data suggest that strains belonging to the STEC O118:[H16] clonal group have a characteristic propensity for acquisition and maintenance of resistance determinants, thus contrasting to STEC belonging to other serotypes.
Detection by PCR-Enzyme-Linked Immunosorbent Assay ofClostridium botulinumin Fish and Environmental Samples from a Coastal Area in Northern France
Authors: Fach Patrick , Perelle Sylvie , Dilasser Françoise , Grout Joël , Dargaignaratz Claire , Botella Lucien , Gourreau Jean-Marie , Carlin Frédéric , Popoff Michel R. , Broussolle Véronique
Published: November 2002

The prevalence ofClostridium botulinumtypes A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10C. botulinumspores per 25 g and 100% of samples inoculated with more than 30C. botulinumspores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence ofC. botulinumin seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2C. botulinummost-probable-number counts, respectively, and is in the low range ofC. botulinumcontamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence ofC. botulinumtype B in fish samples is relatively unusual compared with the high prevalence ofC. botulinumtype E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.
Surface Display of Foreign Epitopes on the
Lactobacillus brevis
S-Layer
Authors: Åvall-Jääskeläinen Silja , Kylä-Nikkilä Kari , Kahala Minna , Miikkulainen-Lahti Terhi , Palva Airi
Published: November 2002

So far, the inability to establish viable
Lactobacillus
surface layer (S-layer) null mutants has hampered the biotechnological applications of
Lactobacillus
S-layers. In this study, we demonstrate the utilization of
Lactobacillus brevis
S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system,
L. brevis
strains producing chimeric S-layers were obtained after testing of four insertion sites in the
slpA
gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-
myc
proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for
L. brevis
and used for the replacement of the wild-type
slpA
gene with the
slpA
-c-
myc
construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a
Lactobacillus
S-layer while still maintaining the S-layer lattice structure.
Isolation and Characterization of Metal-Reducing
Thermoanaerobacter
Strains from Deep Subsurface Environments of the Piceance Basin, Colorado
Authors: Roh Yul , Liu Shi V. , Li Guangshan , Huang Heshu , Phelps Tommy J. , Zhou Jizhong
Published: November 2002

Five bacterial strains were isolated from anaerobic enrichment cultures that had originated from inoculations with samples collected from the deep subsurface environments of the millions-of-years-old, geologically and hydrologically isolated Piceance Basin in Colorado. Small-subunit rRNA gene-based analyses indicated that all of these bacteria were closely related to
Thermoanaerobacter ethanolicus
, with similarities of 99.4 to 99.5%. Three isolates (X513, X514, and X561) from the five bacterial strains were used to examine physiological characteristics. These thermophilic bacteria were able to use acetate, glucose, hydrogen, lactate, pyruvate, succinate, and xylose as electron donors while reducing Fe(III), cobalt(III), chromium(VI), manganese(IV), and uranium(VI) at 60°C. One of the isolates (X514) was also able to utilize hydrogen as an electron donor for Fe(III) reduction. These bacteria exhibited diverse mineral precipitation capabilities, including the formation of magnetite (Fe
3
O
4
), siderite (FeCO
3
), rhodochrosite (MnCO
3
), and uraninite (UO
2
). The gas composition of the incubation headspace and the ionic composition of the incubation medium exerted profound influences on the types of minerals formed. The susceptibility of the thermophilic Fe(III)-reducing cultures to metabolic inhibitors specific for ferric reductase, hydrogenase, and electron transport indicated that iron reduction by these bacteria is an enzymatic process.
The Genus
Caedibacter
Comprises Endosymbionts of
Paramecium
spp. Related to the
Rickettsiales
(
Alphaproteobacteria
) and to
Francisella tularensis
(
Gammaproteobacteria
)
Authors: Beier Cora L. , Horn Matthias , Michel Rolf , Schweikert Michael , Görtz Hans-Dieter , Wagner Michael
Published: November 2002

Obligate bacterial endosymbionts of paramecia able to form refractile inclusion bodies (R bodies), thereby conferring a killer trait upon their ciliate hosts, have traditionally been grouped into the genus
Caedibacter
. Of the six species described to date, only the
Paramecium caudatum
symbiont
Caedibacter caryophilus
has been phylogenetically characterized by its 16S rRNA gene sequence, and it was found to be a member of the
Alphaproteobacteria
related to the
Rickettsiales
. In this study, the
Caedibacter taeniospiralis
type strain, an R-body-producing cytoplasmatic symbiont of
Paramecium tetraurelia
strain 51k, was investigated by comparative 16S rRNA sequence analysis and fluorescence in situ hybridization with specific oligonucleotide probes.
C. taeniospiralis
is not closely related to
C. caryophilus
(80% 16S rRNA sequence similarity) but forms a novel evolutionary lineage within the
Gammaproteobacteria
with the family
Francisellaceae
as a sister group (87% 16S rRNA sequence similarity). These findings demonstrate that the genus
Caedibacter
is polyphyletic and comprises at least two phylogenetically different bacterial species belonging to two different classes of the
Proteobacteria
. Comparative phylogenetic analysis of
C. caryophilus
, five closely related
Acanthamoeba
endosymbionts (including one previously uncharacterized amoebal symbiont identified in this study), and their hosts suggests that the progenitor of the alphaproteobacterial
C. caryophilus
lived within acanthamoebae prior to the infection of paramecia.
The Biodiversity of Lactic Acid Bacteria in Greek Traditional Wheat Sourdoughs Is Reflected in Both Composition and Metabolite Formation
Authors: De Vuyst Luc , Schrijvers Vincent , Paramithiotis Spiros , Hoste Bart , Vancanneyt Marc , Swings Jean , Kalantzopoulos George , Tsakalidou Effie , Messens Winy
Published: November 2002

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the speciesLactobacillus sanfranciscensis,Lactobacillus brevis,Lactobacillus paralimentarius, andWeissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the speciesW. cibariahave not been isolated from sourdoughs previously. NoLactobacillus pontisorLactobacillus panisstrains were found. AnL. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested exceptL. sanfranciscensis. Two of theL. paralimentariusisolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.
Monitoring Changing Toxigenicity of a Cyanobacterial Bloom by Molecular Methods
Authors: Baker Judith A. , Entsch Barrie , Neilan Brett A. , McKay David B.
Published: November 2002

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of
Microcystis aeruginosa
that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the β and α subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of
Anabaena circinalis
strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of
Anabaena
and
Microcystis
spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.
Sequencing-Independent Method To Generate Oligonucleotide Probes Targeting a Variable Region in Bacterial 16S rRNA by PCR with Detachable Primers
Authors: Bertilsson Stefan , Cavanaugh Colleen M. , Polz Martin F.
Published: November 2002

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3′ end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of
Vibrio
. The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.
Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan
Authors: Sutherland Tara D. , Horne Irene , Russell Robyn J. , Oakeshott John G.
Published: November 2002

The gram-positive bacterium
Mycobacterium
sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a
Mycobacterium
-
Escherichia coli
shuttle vector and screened for endosulfan-degrading activity in
Mycobacterium smegmatis
, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (
esd
) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the
M. smegmatis
genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.
Genetic Changes To Optimize Carbon Partitioning between Ethanol and Biosynthesis in EthanologenicEscherichia coli
Authors: Underwood S. A. , Zhou S. , Causey T. B. , Yomano L. P. , Shanmugam K. T. , Ingram L. O.
Published: November 2002

The production of ethanol from xylose by ethanologenicEscherichia colistrain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of nativeE. colialcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.
Multiple
Streptococcus mutans
Genes Are Involved in Biofilm Formation
Authors: Yoshida Akihiro , Kuramitsu Howard K.
Published: November 2002

Streptococcus mutans
has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in
S. mutans
biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to
S. mutans
. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the
Bacillus subtilis lytR
homologue. Another of the biofilm-defective mutants isolated was a
yulF
homologue, which encodes a hypothetical protein of
B. subtilis
whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the
comB
gene required for competence. We therefore have constructed and examined
comACDE
null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the
sgp
and
dgk
genes may play important roles in
S. mutans
biofilm formation.
Effect of Different NADH Oxidase Levels on Glucose Metabolism by
Lactococcus lactis
: Kinetics of Intracellular Metabolite Pools Determined by In Vivo Nuclear Magnetic Resonance
Authors: Neves Ana Rute , Ramos Ana , Costa Helena , van Swam Iris I. , Hugenholtz Jeroen , Kleerebezem Michiel , de Vos Willem , Santos Helena
Published: November 2002

Three isogenic strains of
Lactococcus lactis
with different levels of H
2
O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain
L. lactis
MG1363, a NOX
−
strain harboring a deletion of the gene coding for H
2
O-forming NADH oxidase, and a NOX
+
strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using
13
C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P
2
] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P
2
depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX
−
strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX
+
strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX
−
and parent strains; in the NOX
+
strain the carbon flux was diverted to the production of α-acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.
Fingerprinting Closely Related
Xanthomonas
Pathovars with Random Nonamer Oligonucleotide Microarrays
Authors: Kingsley Mark T. , Straub Timothy M. , Call Douglas R. , Daly Don S. , Wunschel Sharon C. , Chandler Darrell P.
Published: November 2002

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related
Xanthomonas
pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of
X. oryzae
strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.
Identification and Characterization of anN-Acylhomoserine Lactone-Dependent Quorum-Sensing System inPseudomonas putidaStrain IsoF
Authors: Steidle Anette , Allesen-Holm Marie , Riedel Kathrin , Berg Gabriele , Givskov Michael , Molin Søren , Eberl Leo
Published: November 2002

Recent reports have shown that several strains ofPseudomonas putidaproduceN-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promotingP. putidastrain IsoF that, when present intrans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes:ppuIandppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region betweenppuIandppuRwith significant homology torsaLfromPseudomonas aeruginosa, and a gene, designatedppuA,present upstream ofppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptionalppuA::luxABfusion we demonstrate that expression ofppuAis AHL dependent. Furthermore, transcription of the AHL synthaseppuIis shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking theppugene cluster indicated that the four genes form an island in thesuhB-PA3819 intergenic region of the currently sequencedP. putidastrain KT2440. Moreover, we provide evidence that theppugenes are not present in other AHL-producingP. putidastrains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both appuIand appuAmutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development.
Sensitivity of
Listeria monocytogenes
to Sanitizers Used in the Meat Processing Industry
Authors: Romanova Nadya , Favrin Stacy , Griffiths Mansel W.
Published: November 2002

Nineteen
Listeria monocytogenes
strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of
L. monocytogenes
strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the
mdrL
gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.
Sakacin G, a New Type of Antilisterial Bacteriocin
Authors: Simon L. , Fremaux C. , Cenatiempo Y. , Berjeaud J. M.
Published: November 2002

Sakacin G is a 37-amino-acid-residue-long class IIa bacteriocin produced by
Lactobacillus sake
2512, which is encoded by the duplicated structural genes
skgA1
and
skgA2
. Sakacin G appears to be unique and seems to be an intermediate between pediocin-like bacteriocins, according to its double-disulfide bridges required for antimicrobial activity, and mesentericin-like bacteriocins in terms of sequence homologies, inhibition spectrum, and specific activity.
Competitive PCR for Quantitation of a
Cytophaga-Flexibacter-Bacteroides
Phylum Bacterium Associated with the
Tuber borchii
Vittad. Mycelium
Authors: Barbieri Elena , Riccioni Giulia , Pisano Anna , Sisti Davide , Zeppa Sabrina , Agostini Deborah , Stocchi Vilberto
Published: November 2002

An uncultured bacterium associated with the ectomycorrhizal fungus
Tuber borchii
Vittad. was identified as a novel member of the
Cytophaga-Flexibacter-Bacteroides
group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10
6
cells per 30-day-old
T. borchii
mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.
Characterization of
rpoB
Mutations in Rifampin-Resistant Clinical Isolates of
Mycobacterium tuberculosis
from Turkey by DNA Sequencing and Line Probe Assay
Authors: Cavusoglu Cengiz , Hilmioglu Suleyha , Guneri Sevinc , Bilgic Altinay
Published: November 2002

Mutations in an 81-bp region of the
rpoB
gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of
Mycobacterium tuberculosis
isolated in Turkey. Fourteen different
rpoB
alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.
Differences in Virulence Factors among Clinical Isolates of
Escherichia coli
Causing Cystitis and Pyelonephritis in Women and Prostatitis in Men
Authors: Ruiz Joaquim , Simon Karine , Horcajada Juan P. , Velasco Maria , Barranco Margarita , Roig Gloria , Moreno-Martínez Antonio , Martínez Jose A. , Jiménez de Anta Teresa , Mensa Josep , Vila Jordi
Published: November 2002

Differences in the presence of nine urovirulence factors among clinical isolates of
Escherichia coli
causing cystitis and pyelonephritis in women and prostatitis in men have been studied. Hemolysin and necrotizing factor type 1 occur significantly more frequently among isolates causing prostatitis than among those causing cystitis (
P
< 0.0001) or pyelonephritis (
P
< 0.005). Moreover, the
papGIII
gene occurred more frequently in
E. coli
isolates associated with prostatitis (27%) than in those associated with pyelonephritis (9%) (
P
< 0.05). Genes encoding aerobactin and PapC occurred significantly less frequently in isolates causing cystitis than in those causing prostatitis (
P
< 0.01 and
P
< 0.0001, respectively) and pyelonephritis (
P
< 0.01 and
P
< 0.0001, respectively). No differences in the presence of Sat or type 1 fimbriae were found. Finally, AAFII and Bfp fimbriae are no longer considered uropathogenic virulence factors since they were not found in any of the strains analyzed. Overall, the results showed that clinical isolates producing prostatitis need greater virulence than isolates producing pyelonephritis in women or, in particular, cystitis in women (
P
< 0.05). Overall, the results suggest that clinical isolates producing prostatitis are more virulent that those producing pyelonephritis or cystitis in women.
Evaluation of the Makromed Dipstick Assay versus PCR for Diagnosis of
Plasmodium falciparum
Malaria in Returned Travelers
Authors: Richardson David C. , Ciach Michele , Zhong Kathleen J. Y. , Crandall Ian , Kain Kevin C.
Published: November 2002

Microscopy has been the traditional reference standard for malaria diagnosis. However, difficulty in maintaining the required technical skills, particularly in areas where malaria is not endemic, has prompted the development of rapid nonmicroscopic diagnostic assays based on the detection of malaria parasite antigen in whole blood. In this study, we evaluated the performance of one such device, the Makromed dipstick test, blindly compared to PCR and microscopy for the diagnosis of
Plasmodium falciparum
malaria in 200 febrile returned travelers. The Makromed assay detects the presence of
P. falciparum
-specific histidine-rich protein II with an antigen capture immunochromatographic strip format. Compared to PCR as the reference standard, the dipstick assay had a sensitivity of 97.0% and a specificity of 96.0%. The positive and negative predictive values were 81.2% and 99.5%, respectively. Rapid malaria diagnostic devices may provide a useful diagnostic adjunct in a clinical setting.
Molecular Identification and Typing of Enteroviruses Isolated from Clinical Specimens
Authors: Manzara Stefania , Muscillo Michele , La Rosa Giuseppina , Marianelli Cinzia , Cattani Paola , Fadda Giovanni
Published: November 2002

Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5′ noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.
Typing of
Neisseria gonorrhoeae
Reveals Rapid Reinfection in Rural South Africa
Authors: Moodley Prashini , Martin Iona M. C. , Ison Catherine A. , Sturm A. Willem
Published: November 2002

A recent study afforded us the opportunity to collect pre- and post-treatment isolates of
Neisseria gonorrhoeae
from women who supposedly failed to eradicate the organism when tested 8 to 10 days following treatment with a single, directly observed 250-mg dose of ciprofloxacin. In an attempt to differentiate true treatment failure from reinfection, we determined the ciprofloxacin MICs and performed auxotyping, serotyping, and opa typing of the pre- and post-treatment isolates. Paired isolates of
N. gonorrhoeae
were obtained from seven different women, despite susceptibility of the initial isolates to ciprofloxacin. Six of seven patients were infected with gonococcal isolates that differed significantly from their primary isolate. These most probably represent reinfection with a different strain, which could originate from the same partner infected with multiple strains or reinfected with a new strain or from a different partner. The susceptibility to ciprofloxacin of all isolates makes the possibility of multiple strains in the patient unlikely. The diversity of the isolates within the pairs therefore suggests rapid reinfection within the partnerships.
Campylobacter jejuniin Black-Headed Gulls (Larus ridibundus): Prevalence, Genotypes, and Influence onC. jejuniEpidemiology
Authors: Broman T. , Palmgren H. , Bergström S. , Sellin M. , Waldenström J. , Danielsson-Tham M.-L. , Olsen B.
Published: November 2002

Campylobacteriosis is a zoonotic disease in which birds have been suggested to play an important role as a reservoir. We investigated the prevalence ofCampylobacter jejunisubsp. jejuniin black-headed gulls (Larus ridibundus) in southern Sweden with the aim of examining the nature ofC. jejuniinfection in this bird species. Birds were sampled in four sampling series each year during 1999 (n= 419) and 2000 (n= 365). Longitudinally sampledC. jejuniisolates from individual gulls were subjected to macrorestriction profiling (MRP) by pulsed-field gel electrophoresis to investigate the genotypical stability during the natural course of infection. Furthermore, a subset (n= 76) of black-headed gull isolates was compared to isolates from broiler chickens (n= 38) and humans (n= 56) originating from the same geographic area. We found a pronounced seasonal variation inC. jejunicarriage, with the highest rates found in late autumn. MRP similarities were higher between isolates of human and broiler chicken origin, than between those of wild bird origin and either of the other two hosts. However, identical MRPs were found in two gull isolates and one human isolate after digestion with two restriction enzymes, strongly indicating that they may have been colonized by the same clone ofC. jejuni. The MRPs most prevalent in gull isolates did not occur among isolates from humans and broiler chickens, suggesting the existence of a subpopulation ofC. jejuniadapted to species-specific colonization or environmental survival.
Characterization of Shiga Toxin-Producing
Escherichia coli
O157:H7 Isolated in Italy and in France
Authors: Giammanco Giovanni M. , Pignato Sarina , Grimont Francine , Grimont Patrick A. D. , Caprioli Alfredo , Morabito Stefano , Giammanco Giuseppe
Published: November 2002

Twenty-one
Escherichia coli
O157:H7 strains isolated in northern Italy from sporadic cases of hemolytic-uremic syndrome and from cattle and food were characterized by virulence gene analysis, pulsed-field gel electrophoresis (PFGE) of
Xba
I-digested DNA, enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR), and antibiotic resistance patterns and compared to 18 strains isolated in France from human cases of diarrhea, cattle, and the environment. Strains isolated in Sicily (southern Italy) from a local farm (one strain) and from calves just imported from France (11 strains) and Spain (six strains) were also typed. Whereas the
eae
and
hlyA
genes were always detected, Shiga toxin gene (
stx
) analysis showed some differences related to geographic areas. Isolates from northern Italy showed a high frequency of
stx
1
and
stx
2
, while strains isolated in France and from French and Spanish calves imported to Sicily more frequently possessed the
stx
2c
gene. The majority of the strains isolated in northern Italy were also resistant to one or more antibiotics, while most of the strains isolated in France and Sicily were fully susceptible. ERIC-PCR analysis was not able to differentiate the strains. PFGE typing after
Xba
I DNA digestion produced a total of 54 distinct restriction endonuclease digestion profiles (REDPs) among the 57 strains. Phylogenetic analysis was unable to cluster REDPs according to geographic origin. All epidemiologically related isolates showed either identical or ≥91% similar REDPs. Our findings suggest a peculiar circulation of antibiotic-resistant, genetically unrelated strains in northern Italy.