bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling
Authors: Buttner Mark P. , Cruz Patricia , Stetzenbach Linda D. , Klima-Comba Amy K. , Stevens Vanessa L. , Emanuel Peter A. 
Published: December 2004

ABSTRACT

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of
Bacillus atrophaeus
spores, a simulant for
Bacillus anthracis
, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m
2
) sampled with the BiSKit resulted in concentrations of
B. atrophaeus
in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of
B. atrophaeus
on metal surfaces were 42 ± 5.8 CFU/m
2
for wet sampling and 100.5 ± 10.2 CFU/m
2
for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.
Sulfate-Reducing Bacteria in Tubes Constructed by the Marine Infaunal Polychaete
Diopatra cuprea
Authors: Matsui George Y. , Ringelberg David B. , Lovell Charles R. 
Published: December 2004

ABSTRACT

Marine infaunal burrows and tubes greatly enhance solute transport between sediments and the overlying water column and are sites of elevated microbial activity. Biotic and abiotic controls of the compositions and activities of burrow and tube microbial communities are poorly understood. The microbial communities in tubes of the marine infaunal polychaete
Diopatria cuprea
collected from two different sediment habitats were examined. The bacterial communities in the tubes from a sandy sediment differed from those in the tubes from a muddy sediment. The difference in community structure also extended to the sulfate-reducing bacterial (SRB) assemblage, although it was not as pronounced for this functional group of species. PCR-amplified 16S rRNA gene sequences recovered from
Diopatra
tube SRB by clonal library construction and screening were all related to the family
Desulfobacteriaceae
. This finding was supported by phospholipid fatty acid analysis and by hybridization of 16S rRNA probes specific for members of the genera
Desulfosarcina
,
Desulfobacter
,
Desulfobacterium
,
Desulfobotulus
,
Desulfococcus
, and
Desulfovibrio
and some members of the genera
Desulfomonas
,
Desulfuromonas
, and
Desulfomicrobium
with 16S rRNA gene sequences resolved by denaturing gradient gel electrophoresis. Two of six SRB clones from the clone library were not detected in tubes from the sandy sediment. The habitat in which the
D. cuprea
tubes were constructed had a strong influence on the tube bacterial community as a whole, as well as on the SRB assemblage.
Bacterial Life and Dinitrogen Fixation at a Gypsum Rock
Authors: Boison Gudrun , Mergel Alexander , Jolkver Helena , Bothe Hermann 
Published: December 2004

ABSTRACT

The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of
Chroococcidiopsis
spp. The organisms of the shards expressed nitrogenase activity (C
2
H
2
reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of
Chroococcidiopsis
sp. strain PCC7203, C
2
H
2
reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (
nifH
) indicated that
Chroococcidiopsis
possesses the alternative molybdenum nitrogenase 2, expressed in
Anabaena variabilis
only under reduced O
2
tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O
2
tension that allow N
2
fixation to proceed in the unicellular
Chroococcidiopsis
at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of
nifH
sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N
2
under microaerobic conditions only.
Characterization of
Lactobacillus coryniformis
DSM 20001
T
Surface Protein Cpf Mediating Coaggregation with and Aggregation among Pathogens
Authors: Schachtsiek Martina , Hammes Walter P. , Hertel Christian 
Published: December 2004

ABSTRACT

Phenotypic characterization of aggregation phenotypes of
Lactobacillus coryniformis
revealed that strain DSM 20001
T
coaggregated with
Escherichia coli
K88,
Campylobacter coli
, and
Campylobacter jejuni
but not with other human pathogens. In addition, cells of these pathogens aggregated in the presence of the spent culture supernatant (SCS) of strain DSM 20001
T
. Cells of
E. coli
K88 remained viable in the coaggregates and aggregates for up to 24 h. Both coaggregation and aggregation (co/aggregation) occurred at pH 3.5 to 7.5 and was sensitive to heat (85°C for 15 min) and proteinase K. The co/aggregation-promoting factor (Cpf) was purified, and the gene was identified by PCR with degenerate primers derived from internal amino acid sequences. The
cpf
gene encoded a 19.9-kDa preprotein with a
sec
-dependent leader and an isoelectric point of 4.4. The amino acid sequence had no significant similarity to proteins with known functions. Northern analysis revealed not only major transcription from the promoter of
cpf
but also major transcription from the promoter of the preceding insertion element, IS
Lco1
belonging to the IS
3
family. Recombinant Cpf produced in
E. coli
mediated aggregation of pathogens comparable to the aggregation obtained with purified Cpf or SCS of strain DSM 20001
T
. Cpf could be removed from cells of strain DSM 20001
T
by treatment with 5 M LiCl and could be subsequently reattached to the cell surface by using SCS or recombinant Cpf, which resulted in restoration of the co/aggregation property. These results together with those of the amino acid sequence analysis suggest that Cpf is a novel surface protein of
L. coryniformis
that mediates co/aggregation of some pathogens.
Biosynthetic Gene Cluster of the Herbicide Phosphinothricin Tripeptide from
Streptomyces viridochromogenes
Tü494
Authors: Schwartz Dirk , Berger Susanne , Heinzelmann Eva , Muschko Konstanze , Welzel Kathrin , Wohlleben Wolfgang 
Published: December 2004

ABSTRACT

The antibiotic phosphinothricin tripeptide (PTT) consists of two molecules of
l
-alanine and one molecule of the unusual amino acid phosphinothricin (PT) which are nonribosomally combined. The bioactive compound PT has bactericidal, fungicidal, and herbicidal properties and possesses a C—P—C bond, which is very rare in natural compounds. Previously uncharacterized flanking and middle regions of the PTT biosynthetic gene cluster from
Streptomyces viridochromogenes
Tü494 were isolated and sequenced. The boundaries of the gene cluster were identified by gene inactivation studies. Sequence analysis and homology searches led to the completion of the gene cluster, which consists of 24 genes. Four of these were identified as undescribed genes coding for proteins that are probably involved in uncharacterized early steps of antibiotic biosynthesis or in providing precursors of PTT biosynthesis (phosphoenolpyruvate, acetyl-coenzyme A, or
l
-alanine). The involvement of the genes
orfM
and
trs
and of the regulatory gene
prpA
in PTT biosynthesis was analyzed by gene inactivation and overexpression, respectively. Insight into the regulation of PTT was gained by determining the transcriptional start sites of the
pmi
and
prpA
genes. A previously undescribed regulatory gene involved in morphological differentiation in streptomycetes was identified outside of the left boundary of the PTT biosynthetic gene cluster.
Streptomyces coelicolor
A3(2) Lacks a Genomic Island Present in the Chromosome of
Streptomyces lividans
66
Authors: Zhou Xiufen , He Xinyi , Li Aiying , Lei Fang , Kieser Tobias , Deng Zixin 
Published: December 2004

ABSTRACT

Streptomyces lividans
ZX1 has become a preferred host for DNA cloning in
Streptomyces
species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the
S. lividans
66 genome, on which the relative positions of a cloned phage φHAU3 resistance (φHAU3
r
) gene and the
dnd
gene cluster were precisely localized. Comparison of
S. lividans
ZX1 and its progenitor 66, as well as the sequenced genome of its close relative,
Streptomyces coelicolor
M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.
Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent In Situ Hybridization
Authors: Yilmaz L. Safak , Noguera Daniel R. 
Published: December 2004

ABSTRACTIn fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (ΔG°overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA ofEscherichia coliwere used to demonstrate that ΔG°overallis a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold ΔG°overallof −13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.
A Group II Intron-Type Open Reading Frame from the Thermophile
Bacillus
(
Geobacillus
)
stearothermophilus
Encodes a Heat-Stable Reverse Transcriptase
Authors: Vellore Jaishree , Moretz Samuel E. , Lampson Bert C. 
Published: December 2004

ABSTRACT

The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated
trt
, from the genome of the thermophilic bacterium
Bacillus
(
Geobacillus
)
stearothermophilus
strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in
Escherichia coli
, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75°C.
Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum
Authors: Netzer Roman , Peters-Wendisch Petra , Eggeling Lothar , Sahm Hermann 
Published: December 2004

ABSTRACTDespite its key position in central metabolism,l-serine does not support the growth ofCorynebacterium glutamicum. Nevertheless, during growth on glucose,l-serine is consumed at rates up to 19.4 ± 4.0 nmol min−1(mg [dry weight])−1, resulting in the complete consumption of 100 mMl-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of13C-labeledl-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton ofl-serine is mainly converted to pyruvate-derived metabolites such asl-alanine. ThesdaAgene was identified in the genome ofC. glutamicum, and overexpression ofsdaAresulted in (i) functionall-serine dehydratase (l-SerDH) activity, and therefore conversion ofl-serine to pyruvate, and (ii) growth of the recombinant strain onl-serine as the single substrate. In contrast, deletion ofsdaAdecreased thel-serine cometabolism rate with glucose by 47% but still resulted in degradation ofl-serine to pyruvate. Cystathionine β-lyase was additionally found to convertl-serine to pyruvate, and the respectivemetCgene was induced 2.4-fold under high internall-serine concentrations. UponsdaAoverexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellularl-serine conversion to pyruvate might occur, although probably the weak affinity ofl-SerDH (apparentKm, 11 mM) prevents substantiall-serine degradation.
Bile-Mediated Aminoglycoside Sensitivity in
Lactobacillus
Species Likely Results from Increased Membrane Permeability Attributable to Cholic Acid
Authors: Elkins Christopher A. , Mullis Lisa B. 
Published: December 2004

ABSTRACT

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain,
Lactobacillus plantarum
WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 μg/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-
3
H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide
m
-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since
L. plantarum
, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.
Seasonal Incidence of Autochthonous Antagonistic
Roseobacter
spp. and
Vibrionaceae
Strains in a Turbot Larva (
Scophthalmus maximus
) Rearing System
Authors: Hjelm Mette , Riaza Ana , Formoso Fernanda , Melchiorsen Jette , Gram Lone 
Published: December 2004

ABSTRACT

Bacteria inhibitory to fish larval pathogenic bacteria were isolated from two turbot larva rearing farms over a 1-year period. Samples were taken from the rearing site, e.g., tank walls, water, and feed for larvae, and bacteria with antagonistic activity against
Vibrio anguillarum
were isolated using a replica plating assay. Approximately 19,000 colonies were replica plated from marine agar plates, and 341 strains were isolated from colonies causing clearing zones in a layer of
V. anguillarum
. When tested in a well diffusion agar assay, 173 strains retained the antibacterial activity against
V. anguillarum
and
Vibrio splendidus
. Biochemical tests identified 132 strains as
Roseobacter
spp. and 31 as
Vibrionaceae
strains. Partial sequencing of the 16S rRNA gene of three strains confirmed the identification as
Roseobacter gallaeciensis. Roseobacter
spp. were especially isolated in the spring and early summer months. Subtyping of the 132
Roseobacter
spp. strains by randomly amplified polymorphic DNA with two primers revealed that the strains formed a very homogeneous group. Hence, it appears that the same subtype was present at both fish farms and persisted during the 1-year survey. This indicates either a common, regular source of the subtype or the possibility that a particular subtype has established itself in some areas of the fish farm. Thirty-one antagonists were identified as
Vibrio
spp., and 18 of these were
V. anguillarum
but not serotype O1 or O2.
Roseobacter
spp. strains were, in particular, isolated from the larval tank walls, and it may be possible to establish an antagonistic, beneficial microflora in the rearing environment of turbot larvae and thereby limit survival of pathogenic bacteria.
Comparison of Proteolytic Activities Produced by Entomopathogenic
Photorhabdus
Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes
Authors: Marokházi Judit , Lengyel Katalin , Pekár Szilvia , Felföldi Gabriella , Patthy András , Gráf László , Fodor András , Venekei István 
Published: December 2004

ABSTRACT

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic
Photorhabdus
bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (∼74, ∼55, ∼54, and ∼37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J.
379:
633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of
Galleria melonella
larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of
Photorhabdu
s, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into
G. melonella
larvae.
Pressure Inactivation of
Bacillus
Endospores
Authors: Margosch Dirk , Gänzle Michael G. , Ehrmann Matthias A. , Vogel Rudi F. 
Published: December 2004

ABSTRACT

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of
Bacillus subtilis
,
B. amyloliquefaciens
, and
B. licheniformis
to treatments with pressure and temperature (200 to 800 MPa and 60 to 80°C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70°C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain
B. subtilis
CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60°C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of
B. amyloliquefaciens
, which form highly pressure-resistant spores.