bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of Six Honeybee Viruses in Diseased Austrian Apiaries
Authors: Berényi Olga , Bakonyi Tamás , Derakhshifar Irmgard , Köglberger Hemma , Nowotny Norbert
Published: April 2006

The occurrence, prevalence, and distribution patterns of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV) were investigated in 90 Austrian honeybee colonies suffering from symptoms of depopulation, sudden collapse, paralysis, or dark coloring by employing reverse transcription-PCR. Infestation with parasites was also recorded. The samples originated from all parts of Austria. The most prevalent virus was DWV, present in 91% of samples, followed by ABPV, SBV, and BQCV (68%, 49%, and 30%, respectively). CBPV was detected in 10% of colonies, while KBV was not present in any sample. In most samples, more than one virus was identified. The distribution pattern of ABPV, BQCV, CBPV, and SBV varied considerably in the different geographic regions investigated, while DWV was widespread in all Austrian federal states. In bees that showed dark coloring and disorientation, CBPV was always detected. Simultaneous infections of DWV and ABPV were most frequently observed in colonies suffering from weakness, depopulation, and sudden collapse. Bees obtained from apparently healthy colonies within the same apiaries showed a similar distribution pattern of viruses; however, the relative virus load was 10 to 126 times lower than in bees from diseased colonies. A limited number of bee samples from surrounding central European countries (Germany, Poland, Hungary, and Slovenia) were also tested for the presence of the above viruses. Variances were found in the distribution of BQCV and SBV.
Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network
Authors: Thomas Vincent , Herrera-Rimann Katia , Blanc Dominique S. , Greub Gilbert
Published: April 2006

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant
Hartmannella vermiformis
isolate harbored both
Legionella pneumophila
and
Bradyrhizobium japonicum
. By using amoebal coculture with axenic
Acanthamoeba castellanii
as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network.
Alphaproteobacteria
(such as
Rhodoplanes
,
Methylobacterium
,
Bradyrhizobium
,
Afipia
, and
Bosea
) were recovered from 30.5% of the samples, mycobacteria (
Mycobacterium gordonae
,
Mycobacterium kansasii
, and
Mycobacterium xenopi
) were recovered from 20.5% of the samples, and
Gammaproteobacteria
(
Legionella
) were recovered from 5.5% of the samples. No
Chlamydia
or
Chlamydia
-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of
Legionella
(
P
< 0.001) and mycobacteria (
P
= 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.
YliH (BssR) and YceP (BssS) Regulate
Escherichia coli
K-12 Biofilm Formation by Influencing Cell Signaling
Authors: Domka Joanna , Lee Jintae , Wood Thomas K.
Published: April 2006

We previously discovered that
yliH
and
yceP
are induced in
Escherichia coli
biofilms (D. Ren, L. A. Bedzyk, S. M. Thomas, R. W. Ye, and T. K. Wood, Appl. Microbiol. Biotechnol.
64:
515-524, 2004). Here, it is shown that deletion of
yceP
(b1060) and
yliH
(b0836) increases biofilm formation in continuous-flow chambers with minimal glucose medium by increasing biofilm mass (240- to 290-fold), surface coverage (16- to 31-fold), and mean thickness (2,800-fold). To determine the genetic basis of the increase in biofilm formation, we examined the differential gene expression profile in biofilms for both the mutants relative to the wild-type strain in rich medium with glucose and found that 372 to 882 genes were induced and that 76 to 337 were repressed consistently >2-fold (
P
≤ 0.05). The increase in biofilm formation was related to differential expression of genes related to stress response (8 to 64 genes) for both mutants, including
rpoS
and
sdiA
. More importantly, 42 to 130 genes related to autoinducer 2 cell signaling were also differentially expressed, including
gadAB
and
flgBCEGHIJLMN
, as well as signaling through indole, since 17 to 26 indole-related genes were differentially expressed, including
phoAER
,
gltBD
,
mtr
(encodes protein for indole import), and
acrEF
(encodes proteins for indole export). Increased biofilm formation in the
yliH
and
yceP
mutants in LB supplemented with 0.2% glucose (LB glu) occurred through a reduction in extracellular and intracellular indole concentrations in both mutants (50- to 140-fold), and the addition of indole to the culture restored the wild-type biofilm phenotype; hence, indole represses biofilms. Additionally, both mutants regulate biofilms through quorum sensing, since deletion of either
yliH
or
yceP
increased extracellular autoinducer 2 concentrations 50-fold when grown in complex medium (most notably in the stationary phase). Both proteins are involved in motility regulation, since YliH (127 amino acids) and YceP (84 amino acids) repressed motility two to sevenfold (
P
≤ 0.05) in LB, and YceP repressed motility sevenfold (
P
≤ 0.05) in LB glu. Heightened motility in the
yceP
mutant occurred, due to increased transcription of the flagella and motility loci, including
fliC
,
motA
, and
qseB
(3- to 86-fold). We propose new names for these two loci:
bssR
for
yliH
and
bssS
for
yceP
, based on the phrase “regulator of biofilm through signal secretion.”
Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth
Authors: Shapir Nir , Cheng Gang , Sadowsky Michael J. , Wackett Lawrence P.
Published: April 2006

TrzF, the allophanate hydrolase from
Enterobacter cloacae
strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α
2
and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from
Pseudomonas
sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant
Escherichia coli
strain containing
atzD
, encoding cyanuric acid hydrolase that produces biuret, and
atzF
grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains.
E. cloacae
strain 99 grows on cyanuric acid with a significant accumulation of biuret.
Indirect Measurement of the Lag Time Distribution of Single Cells of
Listeria innocua
in Food
Authors: D'Arrigo M. , García de Fernando G. D. , Velasco de Diego R. , Ordóñez J. A. , George S. M. , Pin C.
Published: April 2006

The distribution of log counts at a given time during the exponential growth phase of
Listeria innocua
measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55°C for 45 min or at 62°C for 2 min, and an unheated batch was incubated at 4°C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62°C were greater than those of the cells treated at 55°C. Three batches of paté samples were heated at 55°C for 25 min, 62°C for 81 s, or 65°C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15°C. The distribution of the lag times of the cells heated at 55°C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62°C and 65°C, the lag duration was longer and its variance greater.
Identification of Essential Operons with a Rhamnose-Inducible Promoter in
Burkholderia cenocepacia
Authors: Cardona Silvia T. , Mueller Carmen L. , Valvano Miguel A.
Published: April 2006

Scanning of bacterial genomes to identify essential genes is of biological interest, for understanding the basic functions required for life, and of practical interest, for the identification of novel targets for new antimicrobial therapies. In particular, the lack of efficacious antimicrobial treatments for infections caused by the
Burkholderia cepaci
a complex is causing high morbidity and mortality of cystic fibrosis patients and of patients with nosocomial infections. Here, we present a method based on delivery of the tightly regulated rhamnose-inducible promoter

P
rhaB

for identifying essential genes and operons in
Burkholderia cenocepacia
. We demonstrate that different levels of gene expression can be achieved by using two vectors that deliver

P
rhaB

at two different distances from the site of insertion. One of these vectors places

P
rhaB

at the site of transposon insertion, while the other incorporates the enhanced green fluorescent protein gene (e-
gfp
) downstream from

P
rhaB

. This system allows us to identify essential genes and operons in
B. cenocepacia
and provides a new tool for systematically identifying and functionally characterizing essential genes at the genomic level.
Analyses of the Red-Dry-Rough Phenotype of an
Escherichia coli
O157:H7 Strain and Its Role in Biofilm Formation and Resistance to Antibacterial Agents
Authors: Uhlich Gaylen A. , Cooke Peter H. , Solomon Ethan B.
Published: April 2006

In a previous study, we identified Congo red-binding and -nonbinding phase variants of
Escherichia coli
serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of
Salmonella
. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of
Salmonella enterica
serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike
S. enterica
serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (
P
< 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of
E. coli
O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of
Salmonella
.
Overexpression of the 14α-Demethylase Target Gene (
CYP51
) Mediates Fungicide Resistance in
Blumeriella jaapii
Authors: Ma Zhonghua , Proffer Tyre J. , Jacobs Janette L. , Sundin George W.
Published: April 2006

Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14α-demethylase), through increased expression of the
CYP51
gene, or through increased expression of efflux pumps. We found that
CYP51
expression in DMI-resistant (DMI
R
) isolates of the cherry leaf spot pathogen
Blumeriella jaapii
was increased 5- to 12-fold compared to that in DMI-sensitive (DMI
S
) isolates. Analysis of sequences upstream of
CYP51
in 59 DMI
R
isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of
CYP51
were not present in any of 22 DMI
S
isolates examined.
Specific Growth Rate Determines the Sensitivity of
Escherichia coli
to Thermal, UVA, and Solar Disinfection
Authors: Berney Michael , Weilenmann Hans-Ulrich , Ihssen Julian , Bassin Claudio , Egli Thomas
Published: April 2006

Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of
Escherichia coli
MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (
D
= 0.08 h
−1
) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar
T
90
(time until 90% of the population is inactivated) values (
T
90, chemostat
= 2.66 h;
T
90, batch
= 2.62 h), whereas
T
90
for cells growing at a μ of 0.9 h
−1
was 0.2 h. We present evidence that the stress sensitivity of
E. coli
is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that
E. coli rpoS
mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (μ = 1.0 h
−1
) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (μ = 0.3 h
−1
), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.
Poly(3-Hydroxybutyrate) Synthesis by Recombinant
Escherichia coli arcA
Mutants in Microaerobiosis
Authors: Nikel Pablo I. , Pettinari M. Julia , Galvagno Miguel A. , Méndez Beatriz S.
Published: April 2006

We assessed the effects of different
arcA
mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant
Escherichia coli
strains carrying the
pha
synthesis genes from
Azotobacter
sp. strain FA8. The
arcA
mutations used were an internal deletion and the
arcA2
allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the
arcA
deletion mutant reached a PHB content of 27% ± 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the
arcA2
allele, resulting in a PHB content of 35% ± 3%. This strain grew in a simple medium at a specific growth rate of 0.69 ± 0.07 h
−1
, whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 ± 0.06 h
−1
. The results presented here suggest that
arcA
mutations could play a role in heterologous PHB synthesis in microaerobiosis.
Clonal Dissemination ofEscherichia coliO157:H7 Subtypes among Dairy Farms in Northeast Ohio
Authors: Wetzel Amy N. , LeJeune Jeffrey T.
Published: April 2006

To ascertain the extent to which indistinguishable strains ofEscherichia coliO157:H7 are shared between farms, molecular characterization was performed onE. coliO157:H7 isolates recovered during a longitudinal study of 20 dairy farms in northeast Ohio. Of the 20 dairy farms sampled, 16 were located in a primary area and 4 were located in two other distant geographical areas. A total of 92E. coliO157:H7 isolates obtained from bovine fecal samples, water trough sediment samples, free-stall bedding, and wild-bird excreta samples were characterized. Fifty genetic subtypes were observed among the isolates using XbaI and BlnI restriction endonucleases. Most restriction endonuclease digestion profiles (REDPs) were spatially and temporally clustered. However, four REDPs from multiple sources were found to be indistinguishable by pulsed-field gel electrophoresis between four pairs of farms. The geographical distance between farms which shared an indistinguishableE. coliO157:H7 REDP ranged from 9 to 50 km, and the on-farm sources sharing indistinguishable REDPs included cattle and wild bird feces and free-stall bedding. Within the study population,E. coliO157:H7 REDP subtypes were disseminated with considerable frequency among farms in close geographic proximity, and nonbovine sources may contribute to the transmission of this organism between farms.
Oligonucleotide Array for Identification and Detection of
Pythium
Species
Authors: Tambong J. T. , de Cock A. W. A. M. , Tinker N. A. , Lévesque C. A.
Published: April 2006

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of
Pythium
species. All of the species studied, with the exception of
Pythium ostracodes
, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available
Pythium
sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional
Pythium
isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130
Pythium
isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of
Pythium
species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as
Pythium
species could be a major step forward for epidemiological and ecological studies.
Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors MultipleDehalococcoidesStrains
Authors: Ritalahti Kirsti M. , Amos Benjamin K. , Sung Youlboong , Wu Qingzhong , Koenigsberg Stephen S. , Löffler Frank E.
Published: April 2006

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by differentDehalococcoidesorganisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and threeDehalococcoidesreductive dehalogenase (RDase) genes with assigned function (i.e.,tceA,bvcA, andvcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA fromDehalococcoidespure cultures correlated with standard curves obtained for bothBacteria- andDehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment ofDehalococcoidesorganisms. RDase gene probe/primer pairs were specific for theDehalococcoidesstrains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100tceA,bvcA, orvcrAgene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where knownDehalococcoidesstrains were enumerated, the sum of the three RDase genes equaled the totalDehalococcoidescell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted byDehalococcoides-targeted qPCR, totaling 10 to 30% of the totalDehalococcoidescell numbers. Hence, a large number ofDehalococcoidesspp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinatingDehalococcoidescommunity is incomplete.
Molecular Population Genetics and Phenotypic Diversification of Two Populations of the Thermophilic Cyanobacterium
Mastigocladus laminosus
Authors: Miller Scott R. , Purugganan Michael D. , Curtis Stephanie E.
Published: April 2006

We investigated the distributions of genetic and phenotypic variation for two Yellowstone National Park populations of the heterocyst-forming cyanobacterium
Mastigocladus
(
Fischerella
)
laminosus
that exhibit dramatic phenotypic differences as a result of environmental differences in nitrogen availability. One population develops heterocysts and fixes nitrogen in situ in response to a deficiency of combined nitrogen in its environment, whereas the other population does neither due to the availability of a preferred nitrogen source. Slowly evolving molecular markers, including the 16S rRNA gene and the downstream internal transcribed spacer, are identical among all laboratory isolates from both populations but belie considerable genetic and phenotypic diversity. The total nucleotide diversity at six nitrogen metabolism loci was roughly three times greater than that observed for the human global population. The two populations are genetically differentiated, although variation in performance on different nitrogen sources among genotypes could not be explained by local adaptation to available nitrogen in the respective environments. Population genetic models suggest that local adaptation is mutation limited but also that the populations are expected to continue to diverge due to low migratory gene flow.
Characterization of Intestinal Microbiota and Response to Dietary Virginiamycin Supplementation in the Broiler Chicken
Authors: Dumonceaux Tim J. , Hill Janet E. , Hemmingsen Sean M. , Van Kessel Andrew G.
Published: April 2006

The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinctcpn60sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from theLactobacillales(90% of sequences), while the cecum libraries were more diverse and included members of theClostridiales(68%),Lactobacillales(25%), andBacteroidetes(6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.
Characterization of Biofilm Formation by Clinically Relevant Serotypes of Group A Streptococci
Authors: Lembke Cordula , Podbielski Andreas , Hidalgo-Grass Carlos , Jonas Ludwig , Hanski Emanuel , Kreikemeyer Bernd
Published: April 2006

Streptococcus pyogenes
(group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of
S. pyogenes
in vitro biofilm formation, using
Streptococcus epidermidis
as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.
Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression inListeria monocytogenes
Authors: Bron Peter A. , Monk Ian R. , Corr Sinéad C. , Hill Colin , Gahan Cormac G. M.
Published: April 2006

In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression inListeria monocytogenes. pPL2luxis a derivative of the listerial integration vector pPL2 and harbors a syntheticluxABCDEoperon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2luxderivatives in which thesecAandhlyApromoters were translationally fused toluxABCDEand integrated as a single copy into the chromosome ofL. monocytogenesEGD-e. Growth experiments revealed thathlyAwas expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereassecAexpression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2luxas a quantitative reporter system for real-time, noninvasive evaluation of gene expression inL. monocytogenes.
Reduction of Soluble and Insoluble Iron Forms by Membrane Fractions of
Shewanella oneidensis
Grown under Aerobic and Anaerobic Conditions
Authors: Ruebush Shane S. , Brantley Susan L. , Tien Ming
Published: April 2006

The effect of iron substrates and growth conditions on in vitro dissimilatory iron reduction by membrane fractions of
Shewanella oneidensis
MR-1 was characterized. Membrane fractions were separated by sucrose density gradients from cultures grown with O
2
, fumarate, and aqueous ferric citrate as the terminal electron acceptor. Marker enzyme assays and two-dimensional gel electrophoresis demonstrated the high degree of separation between the outer and cytosolic membrane. Protein expression pattern was similar between chelated iron- and fumarate-grown cultures, but dissimilar for oxygen-grown cultures. Formate-dependent ferric reductase activity was assayed with citrate-Fe
3+
, ferrozine-Fe
3+
, and insoluble goethite as electron acceptors. No activity was detected in aerobic cultures. For fumarate and chelated iron-grown cells, the specific activity for the reduction of soluble iron was highest in the cytosolic membrane. The reduction of ferrozine-Fe
3+
was greater than the reduction of citrate-Fe
3+
. With goethite, the specific activity was highest in the total membrane fraction (containing both cytosolic and outer membrane), indicating participation of the outer membrane components in electron flow. Heme protein content and specific activity for iron reduction was highest with chelated iron-grown cultures with no heme proteins in aerobically grown membrane fractions. Western blots showed that CymA, a heme protein involved in iron reduction, expression was also higher in iron-grown cultures compared to fumarate- or aerobic-grown cultures. To study these processes, it is important to use cultures grown with chelated Fe
3+
as the electron acceptor and to assay ferric reductase activity using goethite as the substrate.
Shear Stress, Temperature, and Inoculation Concentration Influence the Adhesion of Water-Stressed
Helicobacter pylori
to Stainless Steel 304 and Polypropylene
Authors: Azevedo N. F. , Pinto A. R. , Reis N. M. , Vieira M. J. , Keevil C. W.
Published: April 2006

Although molecular techniques have identified
Helicobacter pylori
in drinking water-associated biofilms, there is a lack of studies reporting what factors affect the attachment of the bacterium to plumbing materials. Therefore, the adhesion of
H. pylori
suspended in distilled water to stainless steel 304 (SS304) coupons placed on tissue culture plates subjected to different environmental conditions was monitored. The extent of adhesion was evaluated for different water exposure times, using epifluorescence microscopy to count total cell numbers. High shear stresses—estimated through computational fluid dynamics—negatively influenced the adhesion of
H. pylori
to the substrata (
P
< 0.001), a result that was confirmed in similar experiments with polypropylene (
P
< 0.05). However, the temperature and inoculation concentration appeared to have no effect on adhesion (
P
 > 0.05). After 2 hours,
H. pylori
cells appeared to be isolated on the surface of SS304 and were able to form small aggregates with longer exposure times. However, the formation of a three-dimensional structure was only very rarely observed. This study suggests that the detection of the pathogen in well water described by other authors can be related to the increased ability of
H. pylori
to integrate into biofilms under conditions of low shear stress. It will also allow a more rational selection of locations to perform molecular or plate culture analysis for the detection of
H. pylori
in drinking water-associated biofilms.
A New C-Type Lectin Similar to the Human Immunoreceptor DC-SIGN Mediates Symbiont Acquisition by a Marine Nematode
Authors: Bulgheresi Silvia , Schabussova Irma , Chen Tie , Mullin Nicholas P. , Maizels Rick M. , Ott Jörg A.
Published: April 2006

Although thiotrophic symbioses have been intensively studied for the last three decades, nothing is known about the molecular mechanisms of symbiont acquisition. We used the symbiosis between the marine nematode
Laxus oneistus
and sulfur-oxidizing bacteria to study this process. In this association a monolayer of symbionts covers the whole cuticle of the nematode, except its anterior-most region. Here, we identify a novel Ca
2+
-dependent mannose-specific lectin that was exclusively secreted onto the posterior, bacterium-associated region of
L. oneistus
cuticle. A recombinant form of this lectin induced symbiont aggregation in seawater and was able to compete with the native lectin for symbiont binding in vivo. Surprisingly, the carbohydrate recognition domain of this mannose-binding protein was similar both structurally and functionally to a human dendritic cell-specific immunoreceptor. Our results provide a molecular link between bacterial symbionts and host-secreted mucus in a marine symbiosis and suggest conservation in the mechanisms of host-microbe interactions throughout the animal kingdom.
Protein Kinase C (PkcA) of
Aspergillus nidulans
Is Involved in Penicillin Production
Authors: Herrmann Martina , Spröte Petra , Brakhage Axel A.
Published: April 2006

The biosynthesis of the β-lactam antibiotic penicillin in the filamentous fungus
Aspergillus nidulans
is catalyzed by three enzymes that are encoded by the
acvA
,
ipnA
, and
aatA
genes. A variety of
cis
-acting DNA elements and regulatory factors form a complex regulatory network controlling these β-lactam biosynthesis genes. Regulators involved include the CCAAT-binding complex AnCF and AnBH1. AnBH1 acts as a repressor of the penicillin biosynthesis gene
aatA
. Until now, however, little information has been available on the signal transduction cascades leading to the transcription factors. Here we show that inhibition of protein kinase C (Pkc) activity in
A. nidulans
led to cytoplasmic localization of an AnBH1-enhanced green fluorescent protein (EGFP) fusion protein. Computer analysis of the genome and screening of an
A. nidulans
gene library revealed that the fungus possesses two putative Pkc-encoding genes, which we designated
pkcA
and
pkcB
. Only PkcA showed all the characteristic features of fungal Pkc's. Production of
pkcA
antisense RNA in
A. nidulans
led to reduced growth and conidiation in
Aspergillus
minimal medium, while in fermentation medium it led to enhanced expression of an
aatAp-lacZ
gene fusion, reduced pencillin production, and predominantly cytoplasmic localization of AnBH1. These data agree with the finding that inhibition of Pkc activity prevented nuclear localization of AnBH1-EGFP. As a result, repression of
aatA
expression was relieved. The involvement of Pkc in penicillin biosynthesis is also interesting in light of the fact that in the yeast
Saccharomyces cerevisiae
, Pkc plays a major role in maintaining cell integrity.
Succession and Regulation Factors of Small Eukaryote Community Composition in a Lacustrine Ecosystem (Lake Pavin)
Authors: Lepère Cécile , Boucher Delphine , Jardillier Ludwig , Domaizon Isabelle , Debroas Didier
Published: April 2006

The structure and dynamics of small eukaryotes (cells with a diameter less than 5 μm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples.
Spumella
-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.
Acanthocytes of
Stropharia rugosoannulata
Function as a Nematode-Attacking Device
Authors: Luo Hong , Li Xuan , Li Guohong , Pan Yanbo , Zhang Keqin
Published: April 2006

Efficient killing of nematodes by
Stropharia rugosoannulata
Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode
Panagrellus redivivus
Goodey within minutes and to immobilize the pine wilt nematode
Bursaphelenchus xylophilus
(Steiner & Buhrer) Nickle within hours on agar plates. Moreover,
P. redivivus
worms were completely degraded by the fungus within 24 to 48 h. The cultures of
S. rugosoannulata
studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.
Peptidoglycan Hydrolase Fusions Maintain Their Parental Specificities
Authors: Donovan David M. , Dong Shengli , Garrett Wes , Rousseau Geneviève M. , Moineau Sylvain , Pritchard David G.
Published: April 2006

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide.
Staphylococcus aureus
and
Streptococcus agalactiae
are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated
S. agalactiae
bacteriophage B30 endolysins were fused to the mature lysostaphin protein of
Staphylococcus simulans
. Both fusions display lytic specificity for streptococcal pathogens and
S. aureus
. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.
Isolation, Pure Culture, and Characterization of “
Candidatus
Arsenophonus arthropodicus,” an Intracellular Secondary Endosymbiont from the Hippoboscid Louse Fly
Pseudolynchia canariensis
Authors: Dale Colin , Beeton Michael , Harbison Christopher , Jones Tait , Pontes Mauricio
Published: April 2006

Members of the genus
Arsenophonus
comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an
Arsenophonus
endosymbiont from the hippoboscid louse fly
Pseudolynchia canariensis
. We propose provisional nomenclature for this bacterium in the genus
Arsenophonus
as “
Candidatus
Arsenophonus arthropodicus.” Phylogenetic analyses indicate that “
Candidatus
Arsenophonus arthropodicus” is closely related to the
Arsenophonus
endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of
Arsenophonus
in insects. To this end, we describe methods for the culture of “
Candidatus
Arsenophonus arthropodicus” in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.
Analysis of the Thermostability Determinants of Hyperthermophilic Esterase EstE1 Based on Its Predicted Three-Dimensional Structure
Authors: Rhee Jin-Kyu , Kim Do-Yun , Ahn Dae-Gyun , Yun Jung-Hyuk , Jang Seung-Hwan , Shin Hang-Cheol , Cho Hyun-Soo , Pan Jae-Gu , Oh Jong-Won
Published: April 2006

The three-dimensional (3D) structure of the hyperthermophilic esterase EstE1 was constructed by homology modeling using
Archaeoglobus fulgidus
esterase as a reference, and the thermostability-structure relationship was analyzed. Our results verified the predicted 3D structure of EstE1 and identified the ion pair networks and hydrophobic interactions that are critical determinants for the thermostability of EstE1.
Characterization of
Streptococcus thermophilus
Host Range Phage Mutants
Authors: Duplessis Martin , Lévesque Céline M. , Moineau Sylvain
Published: April 2006

To investigate phage-host interactions in
Streptococcus thermophilus
, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn
917
library of phage-sensitive strain
S. thermophilus
SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10
−6
. Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (
orf15
,
orf17
, and
orf18
) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in
S. thermophilus
.
In Vitro and In Vivo Survival and Transit Tolerance of Potentially Probiotic Strains Carried by Artichokes in the Gastrointestinal Tract
Authors: Valerio Francesca , De Bellis Palmira , Lonigro Stella Lisa , Morelli Lorenzo , Visconti Angelo , Lavermicocca Paola
Published: April 2006

The ability of potentially probiotic strains of
Lactobacillus plantarum
and
Lactobacillus paracasei
to survive on artichokes for at least 90 days was shown. The anchorage of bacterial strains to artichokes improved their survival in simulated gastrointestinal digestion.
L. paracasei
IMPC2.1 was further used in an artichoke human feeding study involving four volunteers, and it was shown that the organism could be recovered from stools.
SXT-Related Integrating Conjugative Element in New World
Vibrio cholerae
Authors: Burrus Vincent , Quezada-Calvillo Roberto , Marrero Joeli , Waldor Matthew K.
Published: April 2006

SXT-related integrating conjugative elements (ICEs) became prevalent in Asian
Vibrio cholerae
populations after
V. cholerae
O139 emerged. Here, we describe an SXT-related ICE, ICE
Vch
Mex1, in a Mexican environmental
V. cholerae
isolate. Identification of ICE
Vch
Mex1 represents the first description of an SXT-related ICE in the Western Hemisphere. The significant differences between the SXT and ICE
Vch
Mex1 genomes suggest that these ICEs have evolved independently.
Occurrence of
Vibrio parahaemolyticus
,
V. cholerae
, and
V. vulnificus
in Norwegian Blue Mussels (
Mytilus edulis
)
Authors: Bauer Anette , Østensvik Øyvin , Florvåg Malin , Ørmen Øyvind , Rørvik Liv Marit
Published: April 2006

Vibrio parahaemolyticus
,
V. cholerae
, and
V. vulnificus
were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained
trh
+
V. parahaemolyticus
, while no
tdh
-positive isolates were detected. The
V. cholerae
isolates were non-O:1/non-O:139 serotypes and were
ctxA
negative.
Produce Isolates of the
Escherichia coli
Ont:H52 Serotype That Carry both Shiga Toxin 1 and Stable Toxin Genes
Authors: Monday Steven R. , Keys Christina , Hanson Patricia , Shen Yuelian , Whittam Thomas S. , Feng Peter
Published: April 2006

Produce isolates of the
Escherichia coli
Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.