bluuebunny/crossref_metadata_2025 huggingface dataset:
Study on Analytical Model of Nonlinear Vibration for Elastic Plates With Gaps Under Random Waves
Authors: Shintani Masanori , Hamai Manabu
Published: December 2004

In this paper, an analytical model for the nonlinear elastic-plastic vibration for long plates with gaps subjected to random vibrations is considered. The nonlinear vibration is caused by the collision phenomena between a mass through a gap and plates with thickness of 0.5, 0.6, and 0.8 mm. An elastic perfectly plastic solid material is assumed in some cases, which adds another aspect to the nonlinear behavior of the system. The material characteristic of the steel is assumed to be an elasto-plasticity solid model. A restoring force characteristic is obtained as the nonlinear vibration of a cubic equation for 0.5, 0.6, and 0.8 mm, the thickness of the plates by experiments. Now the analytical model is proposed by the elasto-plasticity solid model. The relation between the displacement and the force is described by a complicated equation. The curve from the analytical model is called a deflection curve. The results by the analytical model are compared with the results by the experimental model. The restoring force characteristics by the analysis agree with those of the experiment. The restoring force characteristics of the analysis are described using cubic equations. The simple analysis model for evaluation of the vibration characteristic of the nonlinear vibration system, which performs collision vibration with gaps, is proposed by elasto-plasticity solid model in this paper. The results of this proposed analytical model agree with the experimental results better than the results of the minimum of error of square.
Failure Analysis of the Pressure Vessel by Stainless Steel: 1Cr18Ni9Ti
Authors: Zhou Junbo , Wang Kuisheng , Gao Liping
Published: December 2004

The corrosion failure of 1Cr18Ni9Ti stainless steel pressure vessel was studied with the aid of metallurgical microscopes, scanning electron microscopes, scanning Auger energy spectra and X-ray diffraction meters. The main causes of the failure included: inter-crystalline corrosion initiated at or near welding position between head and body of cylinder, electrochemical corrosion due to chloride ions in electrolyte and corrosive action formed by oxygen separator and hydrogen separator. Some measures of corrosion resistance and design improvement were proposed.
Mechanisms and Modeling Comparing HB7 and A723 High Strength Pressure Vessel Steels
Authors: Troiano E. , Parker A. P. , Underwood J. H.
Published: December 2004

HB7, an ultra-clean, high strength pressure vessel steel manufactured in France, is compared to A723 steel. This steel, suggested as an improved pressure vessel material is currently being proposed for critical applications, and will likely be used more frequently as design engineers discover its capabilities. This paper includes comparisons of strength, fracture toughness, fatigue properties and composition of the two steels, followed by an in-depth comparison and modeling of environmental cracking resistance, Bauschinger-modified residual stresses and fatigue lives. Results indicate that in all critical areas, with the exception of Bauschinger-reduced residual stress, the HB7 is superior to the A723 steel. Particularly for small amounts of autofrettage, near-bore residual stresses are reduced for HB7 steel compared to those for A723 steel at the same strength level. The greatest improvement of the HB7 over the A723 is in environmental cracking resistance. The HB7, when tested in concentrated sulfuric acid, exhibits five orders of magnitude longer crack incubation times and three orders of magnitude slower crack growth rates, when compared to A723 steel at the same strength level.
Stress and Fracture Analysis of Ceramic Lined, Composite or Steel Jacketed Pressure Vessels
Authors: Underwood John H. , Parker Anthony P.
Published: December 2004

Stress and fracture analysis of ceramic-lined cannon pressure vessels is described, for a Si3N4 or SiC liner and A723 steel or carbon-epoxy jacket and with an initial residual interface pressure between liner and jacket and pressure applied to the bore. Room temperature stresses for a steel jacket over ceramic are similar to those for a carbon-epoxy jacket, but both radial and hoop jacket stresses can exceed typical carbon-epoxy strength values. Elevated temperature liner stresses are reduced for a carbon-epoxy jacket, due to the effective increase in interface pressure caused by differential thermal expansion. Critical crack sizes for brittle fracture are generally very small, albeit larger for Si3N4 than SiC due to lower liner stresses and higher fracture toughness for Si3N4.
A Study of the Residual Stress Distribution in an Autofrettaged, Thick-Walled Cylinder With Cross-Bore
Authors: Hameed Amer , Brown R. D. , Hetherington John
Published: December 2004

It may be necessary to provide a radial opening such as gas evacuator holes, or an opening to operate the unlocking of the bolt mechanism by means of exhaust gases, in a gun barrel, which is a thick walled cylinder. A three dimensional finite element analysis has been performed to evaluate the effect of introducing a radial cross-bore in an autofrettaged thick-walled cylinder. From the analysis of the cross-bored autofrettaged cylinder, it was observed that there is a severe localized change in the residual stress profile in the vicinity of the cross-bore. The residual circumferential stress increases in compression at the bore. Similarly it increases in tension at the outer diameter, thus making the outer diameter more vulnerable to fatigue failure or crack initiation under stresses arising as a result of firing. Analyses were also performed by varying the cross-bore diameter and it was observed that, by increasing the diameter of the radial hole, the residual circumferential stress at the bore reduces, while it increases at the outer diameter, with an increase in the cross bore diameter. The re-pressurization pressure of an autofrettaged cylinder with radial cross-bore was found to be approximately 65 percent less than the actual autofrettage pressure in a particular case discussed in this paper. A comparison is also made with the residual stress field which would result if the cross-bore was machined before autofrettage.
Use of
Pseudomonas putida
EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of
Escherichia coli
Authors: Yang Taek Ho , Pan Jae Gu , Seo Yeon Soo , Rhee Joon Shick
Published: December 2004

The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme β-lactamase (Bla) on the surface of
Escherichia coli
by using the translocator domain of the
Pseudomonas putida
outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of
E. coli
. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.
Design and Production in
Aspergillus niger
of a Chimeric Protein Associating a Fungal Feruloyl Esterase and a Clostridial Dockerin Domain
Authors: Levasseur Anthony , Pagès Sandrine , Fierobe Henri-Pierre , Navarro David , Punt Peter , Belaïch Jean-Pierre , Asther Marcel , Record Eric
Published: December 2004

A chimeric enzyme associating feruloyl esterase A (FAEA) from
Aspergillus niger
and dockerin from
Clostridium thermocellum
was produced in
A. niger
. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from
A. niger
glucoamylase and a dibasic proteolytic processing site (
kex
-
2
) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.
Microarray and Functional Gene Analyses of Sulfate-Reducing Prokaryotes in Low-Sulfate, Acidic Fens Reveal Cooccurrence of Recognized Genera and Novel Lineages
Authors: Loy Alexander , Küsel Kirsten , Lehner Angelika , Drake Harold L. , Wagner Michael
Published: December 2004

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “
Syntrophobacteraceae
” were detected in both fens, while signals specific for the genus
Desulfomonile
were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (
dsrAB
) gene sequences;
dsrAB
sequences from
Desulfobacca
-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel
dsrAB
types that were phylogenetically unrelated to all known SRPs were detected in the fens. These
dsrAB
sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.
Broad-Host-Range Plasmid pJB658 Can Be Used for Industrial-Level Production of a Secreted Host-Toxic Single-Chain Antibody Fragment in
Escherichia coli
Authors: Sletta H. , Nedal A. , Aune T. E. V. , Hellebust H. , Hakvåg S. , Aune R. , Ellingsen T. E. , Valla S. , Brautaset T.
Published: December 2004

In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible
Pm/xylS
promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of
Escherichia coli
.
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

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

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

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

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

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

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

In 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

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

Despite 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

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

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

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

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.
Identification and Functional Analysis of the Gene Encoding Methionine-γ-Lyase in
Brevibacterium linens
Authors: Amarita Felix , Yvon Mireille , Nardi Michele , Chambellon Emilie , Delettre Jerôme , Bonnarme Pascal
Published: December 2004

The enzymatic degradation of
l
-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese.
l
-Methionine-γ-lyase (MGL) can convert
l
-methionine to methanethiol (MTL), α-ketobutyrate, and ammonia. The
mgl
gene encoding MGL was cloned from the type strain
Brevibacterium linens
ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the
mgl
gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that
l
-methionine degradation via γ-elimination is a key step in the formation of VSCs in
B. linens
.
PCR Analysis of the Distribution of Unicellular Cyanobacterial Diazotrophs in the Arabian Sea
Authors: Mazard Sophie L. , Fuller Nicholas J. , Orcutt Karen M. , Bridle Oliver , Scanlan Dave J.
Published: December 2004

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N
2
fixers was developed based on newly derived sequences from
Crocosphaera
sp. strain WH 8501 and
Cyanothece
sp. strains WH 8902 and WH 8904 as well as several previously described sequences of
Cyanothece
sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom
Climacodium frauenfeldianum
. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nübel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol.
63:
3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67°E meridian from Victoria, Seychelles, to Muscat, Oman (0.5°S to 26°N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29°C) oligotrophic subsurface waters between 0 and 7°N, but they were also found at a station north of Oman at 26°N, 56°35′E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29°C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.
Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic Aerobic Populations
Authors: Tiago Igor , Chung Ana Paula , Veríssimo António
Published: December 2004

Heterotrophic populations were isolated and characterized from an alkaline groundwater environment generated by active serpentinization, which results in a Ca(OH)
2
-enriched, extremely diluted groundwater with pH 11.4. One hundred eighty-five strains were isolated in different media at different pH values during two sampling periods. To assess the degree of diversity present in the environment and to select representative strains for further characterization of the populations, we screened the isolates by using random amplified polymorphic DNA-PCR profiles and grouped them based on similarities determined by fatty acid methyl ester analysis. Phenotypic characterization, determinations of G+C content, phylogenetic analyses by direct sequencing of 16S rRNA genes, and determinations of pH tolerance were performed with the selected isolates. Although 38 different populations were identified and characterized, the vast majority of the isolates were gram positive with high G+C contents and were affiliated with three distinct groups, namely, strains closely related to the species
Dietzia natrolimnae
(32% of the isolates), to
Frigoribacterium
/
Clavibacter
lineages (29% of the isolates), and to the type strain of
Microbacterium kitamiense
(20% of the isolates). Other isolates were phylogenetically related to strains of the genera
Agrococcus
,
Leifsonia
,
Kytococcus
,
Janibacter
,
Kocuria
,
Rothia
,
Nesterenkonia
,
Citrococcus
,
Micrococcus
,
Actinomyces
,
Rhodococcus
,
Bacillus
, and
Staphylococcus
. Only five isolates were gram negative: one was related to the
Sphingobacteria
lineage and the other four were related to the
α-Proteobacteria
lineage. Despite the pH of the environment, the vast majority of the populations were alkali tolerant, and only two strains were able to grow at pH 11.
Novel Type of Heme-Dependent Oxygenase Catalyzes Oxidative Cleavage of Rubber (Poly-cis-1,4-Isoprene)
Authors: Braaz Reinhard , Fischer Peter , Jendrossek Dieter
Published: December 2004

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grownXanthomonassp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned fromXanthomonassp., as revealed by determination ofm/zvalues and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2— and —CH2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA[rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.
Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus
Paxillus involutus
Authors: Courbot Mikael , Diez Laurent , Ruotolo Roberta , Chalot Michel , Leroy Pierre
Published: December 2004

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (γ-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and γ-glutamylcysteine contents increased when the ectomycorrhizal fungus
Paxillus involutus
was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.
Shear Rate Moderates Community Diversity in Freshwater Biofilms
Authors: Rickard Alexander H. , McBain Andrew J. , Stead Amy T. , Gilbert Peter
Published: December 2004

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S
−1
) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S
−1
). The intermediate shear rate (122 S
−1
) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S
−1
), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.