bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTTo 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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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.