bluuebunny/crossref_metadata_2025 huggingface dataset:
Genes ofEscherichia coliO157:H7 That Are Involved in High-Pressure Resistance
Authors: Malone Aaron S. , Chung Yoon-Kyung , Yousef Ahmed E.
Published: April 2006

SeventeenEscherichia coliO157:H7 strains were treated with ultrahigh pressure at 500 MPa and 23 ± 2°C for 1 min. This treatment inactivated 0.6 to 3.4 log CFU/ml, depending on the strain. The diversity of these strains was confirmed by pulsed-field gel electrophoresis (PFGE) analysis, and there was no apparent association between PFGE banding patterns and pressure resistance. The pressure-resistant strainE. coliO157:H7 EC-88 (0.6-log decrease) and the pressure-sensitive strain ATCC 35150 (3.4-log decrease) were treated with a sublethal pressure (100 MPa for 15 min at 23 ± 2°C) and subjected to DNA microarray analysis using anE. coliK-12 antisense gene chip. High pressure affected the transcription of many genes involved in a variety of intracellular mechanisms of EC-88, including the stress response, the thiol-disulfide redox system, Fe-S cluster assembly, and spontaneous mutation. Twenty-fourE. coliisogenic pairs with mutations in the genes regulated by the pressure treatment were treated with lethal pressures at 400 MPa and 23 ± 2°C for 5 min. The barotolerance of the mutants relative to that of the wild-type strains helped to explain the results obtained by DNA microarray analysis. This study is the first report to demonstrate that the expression of Fe-S cluster assembly proteins and the fumarate nitrate reductase regulator decreases the resistance to pressure, while sigma factor (RpoE), lipoprotein (NlpI), thioredoxin (TrxA), thioredoxin reductase (TrxB), a trehalose synthesis protein (OtsA), and a DNA-binding protein (Dps) promote barotolerance.
Comparison of Vertical Distributions of Prokaryotic Assemblages in the Anoxic Cariaco Basin and Black Sea by Use of Fluorescence In Situ Hybridization
Authors: Lin Xueju , Wakeham Stuart G. , Putnam Isabell F. , Astor Yrene M. , Scranton Mary I. , Chistoserdov Andrei Y. , Taylor Gordon T.
Published: April 2006

Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria,Crenarchaeota, andEuryarchaeotawere more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances ofArchaeain both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group ICrenarchaeota(median, 5.5%) to prokaryotic communities were significantly higher (P< 0.001;n= 20) than those of group IIEuryarchaeota(median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4′,6′-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select.
Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones
Authors: Iwaki Hiroaki , Wang Shaozhao , Grosse Stephan , Bergeron Hélène , Nagahashi Ayako , Lertvorachon Jittiwud , Yang Jianzhong , Konishi Yasuo , Hasegawa Yoshie , Lau Peter C. K.
Published: April 2006

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed inEscherichia colia new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived fromPseudomonassp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km= 8 μM versusKm= 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11to C15ketones, methyl-substituted C5and C6ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (Km= 5.8 μM) and the highest catalytic efficiency (kcat/Kmratio of 7.2 × 105M−1s−1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E> 200) as well as 99%S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E= 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.
Effects of Preculturing Conditions on Lag Time and Specific Growth Rate of
Enterobacter sakazakii
in Reconstituted Powdered Infant Formula
Authors: Kandhai M. C. , Reij M. W. , Grognou C. , van Schothorst M. , Gorris L. G. M. , Zwietering M. H.
Published: April 2006

Enterobacter sakazakii
can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of
E. sakazakii
in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water,
E. sakazakii
was able to grow at temperatures between 8 and 47°C. The estimated optimal growth temperature was 39.4°C, whereas the optimal specific growth rate was 2.31 h
−1
. The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6°C and 47.6°C, respectively. The estimated lag time varied from 83.3 ± 18.7 h at 10°C to 1.73 ± 0.43 h at 37°C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.
A Suppression Subtractive Hybridization Approach Reveals Niche-Specific Genes That May Be Involved in Predator Avoidance in Marine
Synechococcus
Isolates
Authors: Jones H. , Ostrowski M. , Scanlan D. J.
Published: April 2006

Picocyanobacteria of the genus
Synechococcus
are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains,
Synechococcus
sp. strain WH8103 (clade III) and
Synechococcus
sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.
Specific Bacterial, Archaeal, and Eukaryotic Communities in Tidal-Flat Sediments along a Vertical Profile of Several Meters
Authors: Wilms Reinhard , Sass Henrik , Köpke Beate , Köster Jürgen , Cypionka Heribert , Engelen Bert
Published: April 2006

The subsurface of a tidal-flat sediment was analyzed down to 360 cm in depth by molecular and geochemical methods. A community structure analysis of all three domains of life was performed using domain-specific PCR followed by denaturing gradient gel electrophoresis analysis and sequencing of characteristic bands. The sediment column comprised horizons easily distinguishable by lithology that were deposited in intertidal and salt marsh environments. The pore water profile was characterized by a subsurface sulfate peak at a depth of about 250 cm. Methane and sulfate profiles were opposed, showing increased methane concentrations in the sulfate-free layers. The availability of organic carbon appeared to have the most pronounced effect on the bacterial community composition in deeper sediment layers. In general, the bacterial community was dominated by fermenters and syntrophic bacteria. The depth distribution of methanogenic archaea correlated with the sulfate profile and could be explained by electron donor competition with sulfate-reducing bacteria. Sequences affiliated with the typically hydrogenotrophic
Methanomicrobiales
were present in sulfate-free layers. Archaea belonging to the
Methanosarcinales
that utilize noncompetitive substrates were found along the entire anoxic-sediment column. Primers targeting the eukaryotic 18S rRNA gene revealed the presence of a subset of archaeal sequences in the deeper part of the sediment cores. The phylogenetic distance to other archaeal sequences indicates that these organisms represent a new phylogenetic group, proposed as “tidal-flat cluster 1.” Eukarya were still detectable at 360 cm, even though their diversity decreased with depth. Most of the eukaryotic sequences were distantly related to those of grazers and deposit feeders.
Geobacter lovleyi
sp. nov. Strain SZ, a Novel Metal-Reducing and Tetrachloroethene-Dechlorinating Bacterium
Authors: Sung Youlboong , Fletcher Kelly E. , Ritalahti Kirsti M. , Apkarian Robert P. , Ramos-Hernández Natalia , Sanford Robert A. , Mesbah Noha M. , Löffler Frank E.
Published: April 2006

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-
cis
-1,2-dichloroethene (
cis
-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [
cis
-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min
−1
mg of protein
−1
, respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H
2
was consumed to threshold concentrations of 0.08 ± 0.03 nM, 0.16 ± 0.07 nM, and 0.5 ± 0.06 nM, respectively, and acetate was consumed to 3.0 ± 2.1 nM, 1.2 ± 0.5 nM, and 3.6 ± 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species,
Geobacter lovleyi
sp. nov., with
Geobacter
(formerly
Trichlorobacter
)
thiogenes
as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that
Geobacter lovleyi
belongs to a distinct, dechlorinating clade within the metal-reducing
Geobacter
group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.
Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation
Authors: Ledger T. , Pieper D. H. , González B.
Published: April 2006

Phenoxyalkanoic compounds are used worldwide as herbicides.
Cupriavidus necator
JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using
tfd
functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the
tfdB
I
and
tfdB
II
genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the
tfdB
I
and
tfdB
II
genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB
I
contributes to a significantly higher extent than TfdB
II
. Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the
tfdB
genes enhanced the toxic effect of 2,4-DCP on
C. necator
cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by
C. necator
JMP134 and by a different host,
Burkholderia xenovorans
LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two
tfdB
genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.
Quantitative Real-Time
Legionella
PCR for Environmental Water Samples: Data Interpretation
Authors: Joly Philippe , Falconnet Pierre-Alain , André Janine , Weill Nicole , Reyrolle Monique , Vandenesch François , Maurin Max , Etienne Jerome , Jarraud Sophie
Published: April 2006

Quantitative
Legionella
PCRs targeting the 16S rRNA gene (specific for the genus
Legionella
) and the
mip
gene (specific for the species
Legionella pneumophila
) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of
Legionella
CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10
3
CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with
mip
PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative
Legionella
PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.
Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria
Authors: Bäuerle Bettina , Cokesa Željko , Hofmann Silvia , Rieger Paul-Gerhard
Published: April 2006

Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain
Agrobacterium tumefaciens
BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol.
70:
3941-3947, 2004). In the bacterial strain
Ralstonia
sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation
15:
229-239, 2004). The
ite
gene, encoding the IDS-transforming epimerase, and the genes
icl
B
and
icl
S
, encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In
Agrobacterium tumefaciens
BY6, the
ite
gene was on an approximately 180-kb circular plasmid, whereas the
icl
B
gene was chromosomal like the corresponding
icl
S
gene in
Ralstonia
sp. strain SLRS7. Heterologous expression in
Escherichia coli
and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.
Identification of
Salmonella enterica
Serovar Typhimurium Genes Important for Survival in the Swine Gastric Environment
Authors: Bearson Shawn M. D. , Bearson Bradley L. , Rasmussen Mark A.
Published: April 2006

Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for
Salmonella enterica
serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a
phoP
mutant following 20 min of challenge and no survival for mutants of
rpoS
or
fur
. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the
phoP
,
rpoS
,
fur
, and
pnp
genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.
Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel
Authors: Diaz Patricia I. , Chalmers Natalia I. , Rickard Alexander H. , Kong Colin , Milburn Craig L. , Palmer Robert J. , Kolenbrander Paul E.
Published: April 2006

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by
Streptococcus
spp. belonging to the
Streptococcus oralis
/
Streptococcus mitis
group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were
Actinomyces
,
Gemella
,
Granulicatella
,
Neisseria
,
Prevotella
,
Rothia
, and
Veillonella
. Fluorescence in situ hybridization (FISH) confirmed that the proportion of
Streptococcus
sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only
Streptococcus
spp. but also the rarer
Prevotella
spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.
Toxigenic
Vibrio cholerae
in the Aquatic Environment of Mathbaria, Bangladesh
Authors: Alam Munirul , Sultana Marzia , Nair G. Balakrish , Sack R. Bradley , Sack David A. , Siddique A. K. , Ali Afsar , Huq Anwar , Colwell Rita R.
Published: April 2006

Toxigenic
Vibrio cholerae
, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable
V. cholerae
, even in nonepidemic periods. Biweekly environmental surveillance for
V. cholerae
was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on
V. cholerae
O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight
V. cholerae
O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for
V. cholerae
O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of
V. cholerae
O1 in thin films of exopolysaccharides (biofilm). A similar clumping of
V. cholerae
O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for
V. cholerae
O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.
Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System
Authors: Tal Yossi , Watts Joy E. M. , Schreier Harold J.
Published: April 2006

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While
Planctomycetales
were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox
Planctomycetales
in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.
Overexpression and Characterization of an Iron Storage and DNA-Binding Dps Protein from
Trichodesmium erythraeum
Authors: Castruita M. , Saito M. , Schottel P. C. , Elmegreen L. A. , Myneni S. , Stiefel E. I. , Morel F. M. M.
Published: April 2006

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N
2
-fixing marine cyanobacterium
Trichodesmium erythraeum
. A
dps
gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of
T. erythraeum
. This gene codes for a putative Dps

T. erythraeurm

protein (Dps
tery
) with 69% primary amino acid sequence similarity to
Synechococcus
DpsA. We expressed and purified Dps
tery
, and we found that Dps
tery
, like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps
tery
binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps
tery
indicated that it has an iron core that resembles that of horse spleen ferritin.
Methanosarcina
spp. Drive Vinyl Chloride Dechlorination via Interspecies Hydrogen Transfer
Authors: Heimann Axel C. , Batstone Damien J. , Jakobsen Rasmus
Published: April 2006

Two highly enriched cultures containing
Dehalococcoides
spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by
Methanosaeta
, while the other culture was dominated by
Methanosarcina
, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by
Methanosaeta
had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by
Methanosarcina
displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts.
Methanosarcina
-dominated cultures converted a higher percentage of [2-
14
C]acetate to
14
CO
2
when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H
2
) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H
2
levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H
2
levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H
2
as electron donor, in cultures both with and without BES. These findings all indicate that
Methanosarcina
(but not
Methanosaeta
), while cleaving acetate to methane, simultaneously oxidizes acetate to CO
2
plus H
2
, driving hydrogenotrophic dehalorespiration of VC to ethene by
Dehalococcoides
.
Elevated Shear Stress Protects
Escherichia coli
Cells Adhering to Surfaces via Catch Bonds from Detachment by Soluble Inhibitors
Authors: Nilsson Lina M. , Thomas Wendy E. , Sokurenko Evgeni V. , Vogel Viola
Published: April 2006

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of
Escherichia coli
. Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.
Culture Conditions of
Roseobacter
Strain 27-4 Affect Its Attachment and Biofilm Formation as Quantified by Real-Time PCR
Authors: Bruhn Jesper Bartholin , Haagensen Janus Anders Juul , Bagge-Ravn Dorthe , Gram Lone
Published: April 2006

The fish probiotic bacterium
Roseobacter
strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of
Roseobacter
strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions.
Does Green Light Influence the Fluorescence Properties and Structure of Phototrophic Biofilms?
Authors: Roldán M. , Oliva F. , Gónzalez del Valle M. A. , Saiz-Jimenez C. , Hernández-Mariné M.
Published: April 2006

Artificial illumination can harm works of art by inducing the development of photosynthetic biofilms. With the aim of preventing biodeterioration or esthetic damage to such surfaces, we evaluated and compared the effects of illuminating biofilms formed byGloeothece membranacea(cyanobacteria) andChlorella sorokiniana(Chlorophyta) using exclusively white or green light.
Laboratory-Dependent Bacterial Ecology: a Cautionary Tale
Authors: O'Keefe K. J. , Morales N. M. , Ernstberger H. , Benoit G. , Turner P. E.
Published: April 2006

Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model
Escherichia coli
. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of
E. coli
. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.
Antimicrobial Susceptibility and Characterization ofSalmonellaIsolates from Processed Bison Carcasses
Authors: Li Qiongzhen , Skyberg Jerod A. , Fakhr Mohamed K. , Sherwood Julie S. , Nolan Lisa K. , Logue Catherine M.
Published: April 2006

SeventeenSalmonella entericaserovar Hadar isolates recovered from bison were found to possess a range of virulence genes and resistance to tetracycline, gentamicin, sulfamethoxazole, and streptomycin simultaneously. A 1-kb class 1 integron containing theaadA1gene was identified in all isolates. Pulsed-field gel electrophoresis found that all isolates were closely related, indicating the possibility of cross-contamination during processing.
Safety Assessment of the Oral Cavity Probiotic
Streptococcus salivarius
K12
Authors: Burton Jeremy P. , Wescombe Philip A. , Moore Chris J. , Chilcott Chris N. , Tagg John R.
Published: April 2006

Streptococcus salivarius
is a prominent member of the oral microbiota and has excellent potential for use as a probiotic targeting the oral cavity. In this report we document safety data relating to
S. salivarius
K12, including assessment of its antibiogram, metabolic profiles, and virulence determinants, and we examine the microbial composition of saliva following the dosing of subjects with K12.
Prevalence of
Borrelia burgdorferi
Sensu Lato and
Anaplasmataceae
Members in
Ixodes ricinus
Ticks in Alsace, a Focus of Lyme Borreliosis Endemicity in France
Authors: Ferquel Elisabeth , Garnier Martine , Marie Jérôme , Bernède-Bauduin Claire , Baranton Guy , Pérez-Eid Claudine , Postic Danièle
Published: April 2006

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of
Ixodes ricinus
ticks infected by
Borrelia burgdorferi
sensu lato and
Anaplasmataceae
. The peak density of nymphs infected by
B. burgdorferi
sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m
2
, respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m
2
), where the disease incidence was low. The two main species detected in ticks were
Borrelia afzelii
, more frequent in nymphs, and
Borrelia garinii
, more frequent in adult ticks. The rates of tick infection by
Anaplasma phagocytophilum
were 0.4% and 1.2% in nymphs and adults, respectively.
Use of Semiconductor-Based Oligonucleotide Microarrays for Influenza A Virus Subtype Identification and Sequencing
Authors: Lodes Michael J. , Suciu Dominic , Elliott Mark , Stover Axel G. , Ross Marty , Caraballo Marcelo , Dix Kim , Crye James , Webby Richard J. , Lyon Wanda J. , Danley David L. , McShea Andrew
Published: April 2006

In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.
Evaluation of the IDI-MRSA Assay for Detection of Methicillin-Resistant
Staphylococcus aureus
from Nasal and Rectal Specimens Pooled in a Selective Broth
Authors: Desjardins M. , Guibord Christiane , Lalonde B. , Toye B. , Ramotar K.
Published: April 2006

Rapid detection of methicillin-resistant
Staphylococcus aureus
(MRSA) by PCR can be performed directly from nasal specimens with the IDI-MRSA assay. To improve the efficiency of screening, we evaluated the performance of the IDI-MRSA assay for the detection of MRSA from pooled and unpooled specimens cultured in a selective broth. Of the 287 specimens evaluated, 71 were culture and PCR positive, 203 were culture and PCR negative, 3 were culture positive and PCR negative, 8 were culture negative and PCR positive, and 2 remained inhibited. A methicillin-susceptible
Staphylococcus aureus
isolate was recovered from five of the eight specimens with false-positive PCR results. Compared to the results of culture, the sensitivity, specificity, and negative and positive predictive values of the IDI-MRSA assay for detection of MRSA from broth were 96%, 96%, 90%, and 98%, respectively. Following implementation of the IDI-MRSA assay, PCR-positive broths were subcultured for evaluation of assay performance. Of the 298 IDI-MRSA assay-positive broths, the results for 103 could not be confirmed by culture. A methicillin-susceptible
S. aureus
(MSSA) isolate was recovered from 77 of these 103 broths. Repeat testing by the IDI-MRSA assay directly with the MSSA isolates confirmed the original positive PCR result. The positive predictive value of the IDI-MRSA assay fell from 90% during the evaluation phase to 65% postimplementation. The IDI-MRSA assay performed well for the detection of MRSA from a selective broth compared to the performance of the detection of MRSA from culture. However, because of the burden associated with implementation of infection control precautions, cultures remain essential in confirming positive IDI-MRSA results.
Sequence Variation in the T-Cell Epitopes of thePlasmodium falciparumCircumsporozoite Protein among Field Isolates Is Temporally Stable: a 5-Year Longitudinal Study in Southern Vietnam
Authors: Jalloh Amadu , van Thien Huynh , Ferreira Marcelo U. , Ohashi Jun , Matsuoka Hiroyuki , Kanbe Toshio , Kikuchi Akihiko , Kawamoto Fumihiko
Published: April 2006

In an effort to decipher the nature and extent of antigen polymorphisms of malaria parasites in a setting where malaria is hypomesoendemic, we conducted a 5-year longitudinal study (1998 to 2003) by sequencing the Th2R and Th3R epitopes of the circumsporozoite protein (CSP) of 142Plasmodium falciparumfield isolates from Bao Loc, Vietnam. Samples were collected during the high-transmission season, September through December 1998 (n =43), as well as from July 2000 to August 2001 (n= 34), September 2001 to July 2002 (n = 33), and August 2002 to July 2003 (n =32). Marked sequence diversity was noted during the high-transmission season in 1998, but no significant variation in allele frequencies was observed over the years (χ2 = 70.003, degrees of freedom = 57,P= 0.116). The apparent temporal stability in allele frequency observed in this Bao Loc malaria setting may suggest that polymorphism in the Th2R and Th3R epitopes is not maintained by frequency-dependent immune selection. By including 36 isolates from Flores Island, Indonesia, and 19 isolates from Thaton, Myanmar, we investigated geographical patterns of sequence polymorphism for these epitopes in Southeast Asia; among the characterized isolates, a globally distributed variant appears to be predominant in Vietnam (75 of 142 isolates, or 52.8%) as well as in Myanmar (15 of 19 isolates, or 78.9%) and Indonesia (31 of 36 isolates, or 86.1%). Further analyses involving worldwide CSP sequences revealed distinct regional patterns, a finding which, together with the unique mutations observed here, may suggest a possible role for host or local factors in the generation of sequence diversity in the T-cell epitopes of CSP.
Performance of the Gen-Probe Transmission-Mediated Amplification Research Assay Compared to That of a Multitarget Real-Time PCR for
Mycoplasma genitalium
Detection
Authors: Hardick Justin , Giles Julie , Hardick Andrew , Hsieh Yu-Hsiang , Quinn Thomas , Gaydos Charlotte
Published: April 2006

Mycoplasma genitalium
(MG) can cause nongonococcal urethritis and is potentially associated with urethritis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in duplicate. Urine samples from males (
n
= 286) and self-collected vaginal swabs from females (
n
= 321) were analyzed by MTRT PCR for MG and the Gen-Probe transmission-mediated amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respectively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.
Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of
Chlamydophila pneumoniae
in Respiratory Specimens
Authors: Loens K. , Beck T. , Goossens H. , Ursi D. , Overdijk M. , Sillekens P. , Ieven M.
Published: April 2006

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of
Chlamydophila pneumoniae
16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon.
C. pneumoniae
RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type
C. pneumoniae
RNA and 0.1 inclusion-forming unit (IFU) of
C. pneumoniae
. In spiked respiratory specimens, the sensitivity of the
C. pneumoniae
NASBA assay varied between 0.1 and 1 IFU/100 μl sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.
Effects of Delayed-Entry Conditions on the Recovery and Detection of Microorganisms from BacT/ALERT and BACTEC Blood Culture Bottles
Authors: Sautter R. L. , Bills A. R. , Lang D. L. , Ruschell G. , Heiter B. J. , Bourbeau P. P.
Published: April 2006

Manufacturers generally recommend that blood culture bottles be loaded into instruments within a short time of collection. However, in our experience, delays often occur prior to loading the bottles. We examined the effect of holding bottles under various temperatures (T)—room temperature (RT), 4°C, 37°C, and RT for 2 h following incubation at 37°C (to simulate transit [TR])—and for various holding times of 4, 12, and 24 h. We utilized the BacT/ALERT system with FA and FN bottles and the BACTEC system with Plus (PL) and Lytic 10 (LY) bottles. Standardized inocula and 5 ml of blood were added to each bottle. Fifteen organisms were evaluated based upon expected performance: aerobic (FA and PL), anaerobic (FN and LY 10), and facultative (all bottles). Based upon expected performance, the FA and FN bottles recovered 458 of 468 organisms and 282 of 288 organisms, respectively, whereas the PL and LY bottles recovered 453 of 468 organisms and 257 of 288 organisms, respectively (
P
= <0.001, FN versus LY). There were 3, 11, 21, and 27 false-negative results for bottles held at 4°C, RT, 37°C, and TR, respectively. There were 4, 8, and 50 false-negative results for bottles held for 4, 12, and 24 h, respectively. Our results support holding these four bottle types at 4°C or at RT for up to 24 h and at 37°C for up to 12 h. We propose that manufacturers only need to make claims for “delayed entry” when these bottles are held for more than 24 h at 4°C or at RT or for more than 12 h at 37°C.
Characteristic Signatures of the
lytA
Gene Provide a Basis for Rapid and Reliable Diagnosis of
Streptococcus pneumoniae
Infections
Authors: Llull Daniel , López Rubens , García Ernesto
Published: April 2006

The nucleotide sequences of the
lytA
gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two
Streptococcus pseudopneumoniae
strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the
Streptococcus pneumoniae
strains harbored typical
lytA
alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical
lytA
alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the
lytA
gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true
S. pneumoniae
strains and represents an improved diagnostic tool for the study of pneumococcal carriage.