bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Real-Time PCR Assay for Rapid Detection and Quantification ofAlexandrium minutum(a Dinoflagellate)
Authors: Galluzzi Luca , Penna Antonella , Bertozzini Elena , Vila Magda , Garcés Esther , Magnani Mauro
Published: February 2004

The marine dinoflagellate genusAlexandriumincludes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death.A. minutumis the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of theAlexandriumgenus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification ofA. minutumcells in seawater samples.Alexandriumgenus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2A. minutumsequence and culturedA. minutumcells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containingA. minutumcells, we were also able to estimate the number of cells. Several fixedA. minutumbloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.
Metamorphosis of a Scleractinian Coral in Response to Microbial Biofilms
Authors: Webster Nicole S. , Smith Luke D. , Heyward Andrew J. , Watts Joy E. M. , Webb Richard I. , Blackall Linda L. , Negri Andrew P.
Published: February 2004

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the
Alphaproteobacteria
,
Betaproteobacteria
,
Gammaproteobacteria
, and
Cytophaga
-
Flavobacterium
of
Bacteroidetes
had the largest influence on overall community composition. A low abundance of
Archaea
was detected in almost all biofilms, providing the first report of
Archaea
associated with coral reef biofilms. No differences in the relative densities of each subdivision of
Proteobacteria
were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.
Effects of Temperature and Pressure on Sulfate Reduction and Anaerobic Oxidation of Methane in Hydrothermal Sediments of Guaymas Basin
Authors: Kallmeyer Jens , Boetius Antje
Published: February 2004

Rates of sulfate reduction (SR) and anaerobic oxidation of methane (AOM) in hydrothermal deep-sea sediments from Guaymas Basin were measured at temperatures of 5 to 200°C and pressures of 1 × 10
5
, 2.2 × 10
7
, and 4.5 × 10
7
Pa. A maximum SR of several micromoles per cubic centimeter per day was found at between 60 and 95°C and 2.2 × 10
7
and 4.5 × 10
7
Pa. Maximal AOM was observed at 35 to 90°C but generally accounted for less than 5% of SR.
New Approach for Specific Determination of Antibiotics by Use of Luminescent
Escherichia coli
and Immune Serum
Authors: Vlasova Irina I. , Asrieli Tatyana V. , Gavrilova Elisaveta M. , Danilov Vadim S.
Published: February 2004

This paper describes a possible application of luminescent
Escherichia coli
activated by blood serum for high-sensitivity and high-specificity assays of antibiotics in solutions. Antibiotics inhibited luminescence of a genetically engineered
E. coli
strain; the system sensitivity to some antibiotics grew notably after the cells had been preactivated by blood serum. The highest level of sensitivity (2.8 ± 0.6 ng/ml) of luminescent cells was obtained for aminoglycoside antibiotics (gentamicin and streptomycin). It is feasible to create the specific biosensor for antibiotics on the basis of bioluminescent
E. coli
strains by applying sera containing antibodies against the antibiotic under assay. The presence of antibodies specific for gentamicin in serum affects inhibition of luminescent cells by gentamicin but not inhibition by other antibiotics.
Diversity of Phage Types among Archived Cultures of the Demerec Collection of
Salmonella enterica
serovar Typhimurium Strains
Authors: Rabsch Wolfgang , Helm R. Allen , Eisenstark Abraham
Published: February 2004

The existence of several thousand
Salmonella enterica
serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.
Permanent Colonization by
Lactobacillus casei
Is Hindered by the Low Rate of Cell Division in Mouse Gut
Authors: Lee Y. K. , Ho P. S. , Low C. S. , Arvilommi H. , Salminen S.
Published: February 2004

Long residence times of probiotics in the intestinal tract would prolong their potential beneficial health effects and assist colonization. This study investigated the colonization potential of
Lactobacillus casei
Shirota in mouse intestine by using 5 (and 6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE) for assessment of doubling times in different parts of the intestine. The amounts of intestinal water overlying the surfaces of the duodenum, jejunum, ileum, and colon in BALB/c mice were 34.4 ± 2.9, 58.8 ± 6.8, 21.6 ± 2.2, and 8.0 ± 1.0 mg, respectively. Based on the residual concentrations of cFDA-SE-labeled lactobacilli on intestinal mucosal surfaces, the average half times for the wash-out of lactobacilli fed were estimated at 3.98, 1.55, 1.34, and 2.48 days in the duodenum, jejunum, ileum, and colon, respectively. The average doubling times of the lactobacilli, estimated from the residual fluorescent levels of surface-adhered cells, were 4.10, 4.78, 4.56, and 5.59 days in the duodenum, jejunum, ileum, and colon, respectively. It is estimated that the lactobacilli would have to achieve an average doubling time of 1.03 to 2.04 days to colonize the various sections of the mouse intestinal tract more permanently.
Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of
Mycobacterium tuberculosis
in
Streptomyces lividans
Authors: Lara Martha , Servín-González Luis , Singh Mahavir , Moreno Carlos , Cohen Ingrid , Nimtz Manfred , Espitia Clara
Published: February 2004

The gene encoding the 45/47 kDa glycoprotein (Rv1860) of
Mycobacterium tuberculosis
was expressed in
Streptomyces lividans
under its own promoter and under the thiostrepton-inducible
Streptomyces
promoter

P
tipA

. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean α-
d
-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in
S. lividans
is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.
Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification
Authors: Fodor Barna D. , Kovács Ákos T. , Csáki Róbert , Hunyadi-Gulyás Éva , Klement Éva , Maróti Gergely , Mészáros Lívia S. , Medzihradszky Katalin F. , Rákhely Gábor , Kovács Kornél L.
Published: February 2004

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for
Thiocapsa roseopersicina
,
Rhodobacter capsulatus
, and
Methylococcus capsulatus
by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate
Escherichia coli
host. The vectors were useful in purifying different proteins from
T. roseopersicina
. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC
2
protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.
Cyanobacterial-Type, Heteropentameric, NAD
+
-Reducing NiFe Hydrogenase in the Purple Sulfur Photosynthetic Bacterium
Thiocapsa roseopersicina
Authors: Rákhely Gábor , Kovács Ákos T. , Maróti Gergely , Fodor Barna D. , Csanádi Gyula , Latinovics Dóra , Kovács Kornél L.
Published: February 2004

Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium
Thiocapsa roseopersicina
(
hupSL
and
hynSL
) have recently been isolated and characterized. Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD
+
-reducing NiFe hydrogenase was identified and sequenced. The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH). The five genes were localized on a single transcript according to reverse transcription-PCR experiments. A σ
54
-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon. The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H
2
under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N
2
. In-frame deletion of the
hoxE
gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro. This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes. This is the first example of the presence of a cyanobacterial-type, NAD
+
-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium. The potential physiological implications are discussed.
Effect of Tetracycline on Transfer and Establishment of the Tetracycline-Inducible Conjugative Transposon Tn
916
in the Guts of Gnotobiotic Rats
Authors: Bahl Martin Iain , Sørensen Søren J. , Hansen Lars Hestbjerg , Licht Tine Rask
Published: February 2004

We have investigated the transfer of Tn
916
among strains of
Enterococcus faecalis
OG1 colonizing in the intestines of gnotobiotic rats. This animal model allows a low limit of detection and efficient colonization of the chosen bacteria. The animals continuously received tetracycline in drinking water. A tetracycline-sensitive recipient strain was allowed to colonize the animals before the resistant donor was introduced. The numbers of donors, recipients, and transconjugants in fecal samples and intestinal segments were estimated. The bioavailable amounts of tetracycline in fecal samples and intestinal segments were monitored by using bacterial biosensors carrying a transcriptional fusion of a tetracycline-regulated promoter and a
lacZ
reporter gene. Chromosomal locations of Tn
916
in transconjugants isolated either from the same animal or from different animals were compared by Southern blot analysis. Our results indicated that selection for the resistant phenotype was the major factor causing higher numbers of transconjugants in the presence of tetracycline. Tetracycline-sensitive
E. faecalis
cells colonized the intestine even when the concentrations of tetracycline in feces and intestinal luminal contents exceeded growth-inhibitory concentrations. This suggests the existence of tetracycline-depleted microhabitats in the intestinal environment.
N
2
Fixation by Unicellular Bacterioplankton from the Atlantic and Pacific Oceans: Phylogeny and In Situ Rates
Authors: Falcón Luisa I. , Carpenter Edward J. , Cipriano Frank , Bergman Birgitta , Capone Douglas G.
Published: February 2004

N
2
-fixing proteobacteria (α and γ) and unicellular cyanobacteria are common in both the tropical North Atlantic and Pacific oceans. In near-surface waters proteobacterial
nifH
transcripts were present during both night and day while unicellular cyanobacterial
nifH
transcripts were present during the nighttime only, suggesting separation of N
2
fixation and photosynthesis by unicellular cyanobacteria. Phylogenetic relationships among unicellular cyanobacteria from both oceans were determined after sequencing of a conserved region of 16S ribosomal DNA (rDNA) of cyanobacteria, and results showed that they clustered together, regardless of the ocean of origin. However, sequencing of
nifH
transcripts of unicellular cyanobacteria from both oceans showed that they clustered separately. This suggests that unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific share a common ancestry (16S rDNA) and that potential unicellular N
2
fixers have diverged (
nifH
). N
2
fixation rates for unicellular bacterioplankton (including small cyanobacteria) from both oceans were determined in situ according to the acetylene reduction and
15
N
2
protocols. The results showed that rates of fixation by bacterioplankton can be almost as high as those of fixation by the colonial N
2
-fixing marine cyanobacteria
Trichodesmium
spp. in the tropical North Atlantic but that rates are much lower in the subtropical North Pacific.
Determination of Cu Environments in the CyanobacteriumAnabaena flos-aquaeby X-Ray Absorption Spectroscopy
Authors: Kretschmer X. C. , Meitzner G. , Gardea-Torresdey J. L. , Webb R.
Published: February 2004

Whole cells and peptidoglycan isolated from cell walls of the cyanobacteriumAnabaena flos-aquaewere lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.
Direct Real-Time PCR Quantification of
Campylobacter jejuni
in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification
Authors: Rudi Knut , Høidal Hilde Kristin , Katla Tone , Johansen Birgit Klungseth , Nordal John , Jakobsen Kjetill S.
Published: February 2004

Campylobacter jejuni
is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of
C. jejuni
in poultry is the cecum, with an estimated content of 6 to 8 log
10
CFU/g. If a flock is infected with
C. jejuni
, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for
C. jejuni
obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with
C. jejuni
and through the detection of
C. jejuni
in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log
10
were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were
Campylobacter
positive, whereas 12 were negative. Two of the flocks contained
Campylobacter
species other than
C. jejuni
. There was a large difference in the
C. jejuni
content, ranging from 4 to 8 log
10
CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of
C. jejuni
to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.
Characterization and Development of Two Reporter Gene Systems for
Clostridium acetobutylicum
Authors: Feustel Lothar , Nakotte Stephan , Dürre Peter
Published: February 2004

The use of
lacZ
from
Thermoanaerobacterium thermosulfurigenes
(encoding β-galactosidase) and
lucB
from
Photinus pyralis
(encoding luciferase) as reporter genes in
Clostridium acetobutylicum
was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of
lacZ
revealed a sequence error in the original database entry, which resulted in β-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The
lacZ
reporter data showed that
bdhA
(encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by
sol
(encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and
bdhB
(encoding butanol dehydrogenase B) expression.
adc
(encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between
adc
and
bdhB
(higher) and
bdhA
and the
sol
operon (lower). The
lucB
reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data).
lucB
reporter measurements confirmed the expression pattern of the
sol
and
ptb-buk
(encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the
lacZ
reporter and showed that the expression level from the
ptb
promoter is 59-fold higher than that from the
sol
operon promoter.
New Perspective on Uncultured Bacterial Phylogenetic Division OP11
Authors: Harris J. Kirk , Kelley Scott T. , Pace Norman R.
Published: February 2004

Organisms belonging to the OP11 candidate phylogenetic division of
Bacteria
have been detected only in rRNA-based sequence surveys of environmental samples. Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically. In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs. Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments. Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing. Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1). The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments. The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts.
Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria
Authors: Haddix Pryce L. , Shaw Nancy J. , LeChevallier Mark W.
Published: February 2004

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample.
Pseudomonas fluorescens
P-17 and
Spirillum
sp. strain NOX bacteria were mutagenized with
luxCDABE
operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with
p
-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.
Chromate-Reducing Properties of Soluble Flavoproteins from
Pseudomonas putida
and
Escherichia coli
Authors: Ackerley D. F. , Gonzalez C. F. , Park C. H. , Blake R. , Keyhan M. , Matin A.
Published: February 2004

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from
Pseudomonas putida
) and YieF (from
Escherichia coli
)—were examined. Both are dimers and reduce chromate efficiently to Cr(III) (
k
cat
/

K
m

= ∼2 × 10
4
M
−1
 · s
−1
). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.
Electrotransformation of
Clostridium thermocellum
Authors: Tyurin Michael V. , Desai Sunil G. , Lynd Lee R.
Published: February 2004

Electrotransformation of several strains of
Clostridium thermocellum
was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of
C. thermocellum
with subsequent PCR specific to the
mls
gene on the plasmid, as well as by retransformation of
Escherichia coli
. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10
5
transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the
mls
gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10
4
transformants per μg of plasmid DNA for strain ATCC 27405 and ∼1 × 10
3
transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ∼50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in
C. thermocellum
that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.
Multi-Virulence-Locus Sequence Typing ofListeria monocytogenes
Authors: Zhang Wei , Jayarao Bhushan M. , Knabel Stephen J.
Published: February 2004

A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtypingListeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) ofL. monocytogeneswas selected from a strain collection (n> 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA,inlB, andinlC) and three virulence-associated genes (dal,lisR, andclpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automatedEcoRI-RT and PFGE withApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology ofL. monocytogenes.
Diazotrophic Community Structure and Function in Two Successional Stages of Biological Soil Crusts from the Colorado Plateau and Chihuahuan Desert
Authors: Yeager Chris M. , Kornosky Jennifer L. , Housman David C. , Grote Edmund E. , Belnap Jayne , Kuske Cheryl R.
Published: February 2004

The objective of this study was to characterize the community structure and activity of N
2
-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of
nifH
sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to
Nostoc
spp. and that the composition of N
2
-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of
nifH
sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a
Microcoleus vaginatus
-dominated, poorly developed crust to mature crusts harboring a greater percentage of
Nostoc
and
Scytonema
spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N
2
-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by
Microcoleus vaginatus
.
Occurrence, Structure, and Mobility of Tn
1546
-Like Elements in Environmental Isolates of Vancomycin-Resistant Enterococci
Authors: Guardabassi L. , Dalsgaard A.
Published: February 2004

The occurrence, structure, and mobility of Tn
1546-
like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored

vanA

. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as
Enterococcus faecium
(
n
= 13),
E. casseliflavus
(
n
= 6),
E. mundtii
(
n
= 3),
E. faecalis
(
n
= 3), and
E. durans
(
n
= 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn
1546
-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn
1546
in the prototype strain
E. faecium
BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for
E. casseliflavus
strains. Indistinguishable Tn
1546-
like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to
E. faecium
BM4105 was demonstrated for
E. faecalis
,
E. mundtii
, and
E. durans
. This study indicates that VRE, including species other than
E. faecium
and
E. faecalis
, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn
1546
-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.
Detection and Differentiation of In Vitro-Spiked Bacteria by Real-Time PCR and Melting-Curve Analysis
Authors: Klaschik S. , Lehmann L. E. , Raadts A. , Book M. , Gebel J. , Hoeft A. , Stuber F.
Published: February 2004

We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and plasma. This prototype detection system enables an exact Gram stain classification and, in particular, screening for specific species of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes and melting-curve analysis in a one-run experiment. One strain of every species was tested at a final density of 10
6
CFU/ml. All bacteria examined except
Staphylococcus aureus
and
Staphylococcus epidermidis
could be differentiated successfully;
S. aureus
and
S. epidermidis
could only be classified as “
Staphylococcus
species.” The hands-on time for preparation of the DNA, performance of the PCR, and evaluation of the PCR results was less than 4 h. Nevertheless, this prototype detection system requires more clinical validation.
Antigen Capture Competitive Enzyme-Linked Immunosorbent Assays Using Baculovirus-Expressed Antigens for Diagnosis of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus
Authors: Mecham J. O. , Wilson W. C.
Published: February 2004

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that infect both livestock and wild ruminants. Antigenic cross-reactivity between BTV and EHDV often results in serologic misdiagnosis. Competitive enzyme-linked immunosorbent assays (c-ELISAs) show increased sensitivity and specificity for the identification of these viral diseases; however, the preparation of cell culture-derived viral antigen for these tests is laborious and variable from batch to batch, and the resulting antigen may be infectious. To overcome these problems, the genes coding for a structural protein, VP7, of BTV and EHDV were cloned into baculovirus and the recombinant proteins were expressed in Sf9 cultured insect cells. Recombinant viral proteins released into the baculovirus-infected Sf9 cell culture supernatant were used in antigen capture c-ELISAs (Ag Cap c-ELISA) tests that specifically detected antibody in the serum of cattle experimentally infected with BTV and EHDV. The diagnostic utility of the Ag Cap c-ELISA was demonstrated by comparison with a commercial c-ELISA. The Ag Cap c-ELISA offers the advantages of using an easily produced, easily standardized, noninfectious antigen that does not require further purification or concentration.
DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR
Authors: Yamasaki Hiroshi , Allan James C. , Sato Marcello Otake , Nakao Minoru , Sako Yasuhito , Nakaya Kazuhiro , Qiu Dongchuan , Mamuti Wulamu , Craig Philip S. , Ito Akira
Published: February 2004

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome
c
oxidase subunit 1 gene yielded evident differential products unique for
Taenia saginata
and
Taenia asiatica
and for American/African and Asian genotypes of
Taenia solium
with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9
T. solium
carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a
T. solium
carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to
T. solium
carriers, 827-bp markers were detected by multiplex PCR in all
T. saginata
carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections.
Multicenter Evaluation of the Performance Characteristics of the Bayer VERSANT HCV RNA 3.0 Assay (bDNA)
Authors: Elbeik Tarek , Surtihadi Johan , Destree Mark , Gorlin Jed , Holodniy Mark , Jortani Saeed A. , Kuramoto Ken , Ng Valerie , Valdes Roland , Valsamakis Alexandra , Terrault Norah A.
Published: February 2004

In this multicenter evaluation, the VERSANT HCV RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Tarrytown, N.Y.) was shown to have excellent reproducibility, linearity, and analytical sensitivity across specimen collection matrices (serum, EDTA, ACD-A), and hepatitis C virus (HCV) genotypes 1 to 6. The VERSANT HCV bDNA Assay has a reportable range of 615 to 7,690,000 (7.69 × 106) IU/ml. The total coefficient of variation (CV) ranged from 32.4% at 615 IU/ml to 17% at 6.8 × 106IU/ml. The assay was linear across the reportable range. Analytical specificity of 98.8% was determined by testing 999 specimens from volunteer blood donors. Evaluation of HCV genotypes using RNA transcripts of representative clones of 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, and 6a and patient specimens showed that the largest difference between genotype 1, upon which the assay is standardized, and non-1 genotypes was within 1.5-fold. Testing of potentially interfering endogenous substances and exogenous substances and conditions found no interference in HCV-positive or HCV-negative specimens except for unconjugated bilirubin at concentrations of ≥20 mg/dl and protein at concentrations of ≥9 g/dl. Biological variability was estimated from 29 clinically stable individuals not on HCV therapy who were tested weekly over an 8-week period. The combined estimate of total (biologic plus assay) variability was 0.15 log10standard deviation (CV, 36.1%), a fold change of 2.6. Thus, the observed fold change between any two consecutive HCV RNA measures is expected to be less than 2.6-fold (equivalent to 0.41 log10IU/ml) 95% of the time in clinically stable individuals.
Evaluation of Partial 16S Ribosomal DNA Sequencing for Identification of
Nocardia
Species by Using the MicroSeq 500 System with an Expanded Database
Authors: Cloud Joann L. , Conville Patricia S. , Croft Ann , Harmsen Dag , Witebsky Frank G. , Carroll Karen C.
Published: February 2004

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate
Nocardia
species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5′ 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i) conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii) molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii) when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of
Nocardia
species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens
N. brasiliensis
,
N. cyriacigeorgica
,
N. farcinica
,
N. nova
,
N. otitidiscaviarum
, and
N. veterana
.
Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments
Authors: Leb Victoria , Stöcher Markus , Valentine-Thon Elizabeth , Hölzl Gabriele , Kessler Harald , Stekel Herbert , Berg Jörg
Published: February 2004

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.
Comparison of Mismatch Amplification Mutation Assay with DNA Sequencing for Characterization of Fluoroquinolone Resistance in
Neisseria gonorrhoeae
Authors: Sultan Zafar , Nahar Shamsun , Wretlind Bengt , Lindback Emma , Rahman Motiur
Published: February 2004

A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of
gyrA
at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of
gyrA
in 117
Neisseria gonorrhoeae
isolates (with ciprofloxacin MICs of 0.004 to >32 μg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the
gyrA
genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in
N. gonorrhoeae
.