bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Detection, Identification, and Enumeration of
Escherichia coli
Cells in Municipal Water by Chemiluminescent In Situ Hybridization
Authors: Stender Henrik , Broomer Adam J. , Oliveira Kenneth , Perry-O'Keefe Heather , Hyldig-Nielsen Jens J. , Sage Andrew , Coull James 
Published: July 2002

ABSTRACT

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable
Escherichia coli
cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of
E. coli
were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in
E. coli
16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of
E. coli
. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of
E. coli
and
Pseudomonas aeruginosa
were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting
P. aeruginosa
16S rRNA. The sensitivity and specificity for the detection of
E. coli
by PNA CISH were then determined using 8
E. coli
strains and 17 other bacterial species, including closely related species. No bacterial strains other than
E. coli
and
Shigella
spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of
E. coli
represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.
Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of
Agrobacterium
Biovars 1 and 2
Authors: Mougel Christophe , Cournoyer Benoit , Nesme Xavier 
Published: July 2002

ABSTRACT

Ecology and biodiversity studies of
Agrobacterium
spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K
2
TeO
3
was analyzed and its potential for isolating
Agrobacterium
spp. directly from soil was evaluated. A K
2
TeO
3
concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K
2
TeO
3
-amended medium. The bona fide agrobacterium colonies growing on media amended with K
2
TeO
3
were confirmed to be
Agrobacterium
colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for
Agrobacterium
biovar 1 and related species (
Agrobacterium rubi
and
Agrobacterium fici
) and for
Agrobacterium
biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the
Agrobacterium
-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10
3
to 10
4
agrobacteria · g of dry soil
−1
in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide
Agrobacterium
biovar 1 strains.
Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1
Authors: Liu Yong , Louie Tai Man , Payne Jason , Bohuslavek Jan , Bolton Harvey , Xun Luying 
Published: July 2002

ABSTRACT

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O
2
. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent

K
m

for IDA was 4.0 mM with a
k
cat
of 5.3 s
−1
. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in
Escherichia coli
, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.
In Vivo Regulation of Glutamine Synthetase Activity in the Marine Chlorophyll
b
-Containing Cyanobacterium
Prochlorococcus
sp. Strain PCC 9511 (Oxyphotobacteria)
Authors: El Alaoui Sabah , Diez Jesús , Humanes Lourdes , Toribio Fermı́n , Partensky Frédéric , Garcı́a-Fernández José Manuel 
Published: July 2002

ABSTRACT

The physiological regulation of glutamine synthetase (GS; EC
6.3.1.2
) in the axenic
Prochlorococcus
sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for
Prochlorococcus
growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of
Prochlorococcus
for coping with a light- and nutrient-limited environment.
Study of the Infectivity of Saline-Stored
Campylobacter jejuni
for Day-Old Chicks
Authors: Hald Birthe , Knudsen Katrine , Lind Peter , Madsen Mogens 
Published: July 2002

ABSTRACT

The culturability of three
Campylobacter jejuni
strains and their infectivity for day-old chicks were assessed following storage of the strains in saline. The potential for colonization of chicks was weakened during the storage period and terminated 3 to 4 weeks before the strains became nonculturable. The results from this study suggest that the role of starved and aged but still culturable campylobacters may be diminutive, but even more, that the role of viable but nonculturable stages in campylobacter epidemiology may be negligible. Even high levels of maternally derived anti-campylobacter outer membrane protein serum antibodies in day-old chicks did not protect the chicks from campylobacter colonization.
Analysis of Bacterial Detachment from Substratum Surfaces by the Passage of Air-Liquid Interfaces
Authors: Gómez-Suárez Cristina , Busscher Henk J. , van der Mei Henny C. 
Published: July 2002

ABSTRACT

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (
Streptococcus sobrinus
HG1025,
Streptococcus oralis
J22,
Actinomyces naeslundii
T14V-J1,
Bacteroides fragilis
793E, and
Pseudomonas aeruginosa
974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 × 10
6
cells cm
−2
was reached. For
S. sobrinus
HG1025,
S. oralis
J22, and
A. naeslundii
T14V-J1, the conditioning film consisted of adsorbed salivary components, while for
B. fragilis
793E and
P. aeruginosa
974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with
P. aeruginosa
974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s
−1
), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as “bacterial retention” rather than “bacterial adhesion”.
Survival and Heat Resistance of
Listeria monocytogenes
after Exposure to Alkali and Chlorine
Authors: Taormina P. J. , Beuchat L. R. 
Published: July 2002

ABSTRACT

A strain of
Listeria monocytogenes
isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells of
L. monocytogenes
incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in
L. monocytogenes
may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log
10
CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log
10
CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by
L. monocytogenes
cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of
L. monocytogenes
against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log
10
-unit reductions is not compromised in these cells.
Ecological Aspects of
ntcA
Gene Expression and Its Use as an Indicator of the Nitrogen Status of Marine
Synechococcus
spp
Authors: Lindell Debbie , Post Anton F. 
Published: July 2002

ABSTRACT

Nitrogen nutrition in cyanobacteria is regulated by NtcA, a transcriptional activator that is subject to negative control by ammonium. Using
Synechococcus
sp. strain WH7803 as a model organism, we show that
ntcA
expression was induced when cells were exposed to nitrogen stress but not when they were subjected to phosphorus or iron deprivation. Transcript levels accumulated in cells grown on a variety of inorganic and organic nitrogen sources, with the sole exception of ammonium.
ntcA
transcription was induced when ammonium levels dropped below 1 μM and reached maximal levels within 2 h. Furthermore, the addition of more than 1 μM ammonium led to a rapid decline in
ntcA
mRNA. The negative effect of ammonium was prevented by the addition of
l
-methionine-
d
,
l
-sulfoximine (MSX) and azaserine, inhibitors of ammonium assimilation. Thus, basal
ntcA
transcript levels are indicative of ammonium utilization. Conversely, the highest
ntcA
transcript levels were found in cells lacking a nitrogen source capable of supporting growth. Therefore, maximal
ntcA
expression would indicate nitrogen deprivation. This state of nitrogen deprivation was induced by a 1-h incubation with MSX. The rapid response of
ntcA
gene expression to the addition of ammonium and MSX was used to design a protocol for assessing relative
ntcA
transcript levels in field populations of cyanobacteria, from which their nitrogen status can be inferred.
ntcA
was basally expressed in
Synechococcus
at a nutrient-enriched site at the northern tip of the Gulf of Aqaba, Red Sea. Therefore, these cyanobacteria were not nitrogen stressed, and their nitrogen requirements were met by regenerated nitrogen in the form of ammonium.
The
P450-4
Gene of
Gibberella fujikuroi
Encodes
ent
-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway
Authors: Tudzynski Bettina , Hedden Peter , Carrera Esther , Gaskin Paul 
Published: July 2002

ABSTRACT

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of
Gibberella fujikuroi
; these genes encode the bifunctional
ent
-copalyl diphosphate synthase/
ent
-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (
P450-4
). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a
P450-4
knockout mutant identified
ent
-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of
ent
-kaurene,
ent
-kaurenol, and
ent
-kaurenal was blocked in the transformants, whereas
ent
-kaurenoic acid was metabolized efficiently to GA
4
. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted
ent
-kaurene to
ent
-kaurenoic acid after transformation with
P450-4
. The B1-41a mutant, described as blocked between
ent
-kaurenal and
ent
-kaurenoic acid, was fully complemented by
P450-4
. There is a single nucleotide difference between the sequence of the B1-41a and wild-type
P450-4
alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that
P450-4
encodes a multifunctional
ent
-kaurene oxidase catalyzing all three oxidation steps between
ent
-kaurene and
ent
-kaurenoic acid.
Identification and Characterization of a Chitinase Antigen from
Pseudomonas aeruginosa
Strain 385
Authors: Thompson Suzanne E. , Smith Mark , Wilkinson Mark C. , Peek Keith 
Published: July 2002

ABSTRACT

A chitinase antigen has been identified in
Pseudomonas aeruginosa
strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH
2
-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the
P. aeruginosa
genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The

K
m

and

k
cat

for 4-nitrophenyl-β-
d
-
N,N
′-diacetylchitobiose were 4.28 mM and 1.7 s
−1
respectively, and for 4-nitrophenyl-β-
d
-
N,N
′,
N
′′-triacetylchitotriose, they were 0.48 mM and 0.16 s
−1
respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50°C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.
Toxic Effects of Ag(I) and Hg(II) onCandida albicansandC. maltosa: a Flow Cytometric Evaluation
Authors: Zhang Shangtong , Crow Sidney A. 
Published: July 2002

ABSTRACTThe effects of Ag(I) and Hg(II) on membrane potential and integrity of cells ofCandida albicansandC. maltosawere determined with a flow cytometric procedure that employed an anionic membrane potential-sensitive dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, and a membrane integrity indicator, propidium iodide. The membrane potentials of cells of both species were reduced rapidly within 15 min of exposure to Ag(I). No threshold dose for Hg(II) existed, and cells of both species lost membrane potential gradually in Hg(II) solutions. Cells of both species lost membrane integrity more rapidly in Ag(I) solutions than in Hg(II) solutions. In Ag(I) solutions, the decrease in the numbers of cells recoverable in culture occurred at a rate similar to the rate of cell depolarization and membrane permeabilization. In Hg(II) solutions, loss of cell recoverability preceded the loss of membrane potential and membrane integrity. C. albicans, in contrast toC. maltosa, showed no loss of membrane integrity after exposure to Hg(II) solutions for 1 h. Different rates of binding of Ag(I) and Hg(II) between the two species suggest that the two ions target different primary sites.
Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food
Authors: Franz Charles M. A. P. , Muscholl-Silberhorn Albrecht B. , Yousif Nuha M. K. , Vancanneyt Marc , Swings Jean , Holzapfel Wilhelm H. 
Published: July 2002

ABSTRACT

The incidence of virulence factors among 48
Enterococcus faecium
and 47
Enterococcus faecalis
strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of
E. faecium
strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of
E. faecalis
strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.
Molecular Monitoring of Succession of Bacterial Communities in Human Neonates
Authors: Favier Christine F. , Vaughan Elaine E. , De Vos Willem M. , Akkermans Antoon D. L. 
Published: July 2002

ABSTRACTThe establishment of bacterial communities in two healthy babies was examined for more than the first 10 months of life by monitoring 16S ribosomal DNA (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE) and by analyzing the sequences of the major ribotypes. DGGE profiles of the dominant populations in the intestines of the infants were obtained by analyzing daily or weekly fecal samples. After delivery, the germfree infant gastrointestinal tracts were rapidly colonized, and the succession of bacteria in each ecosystem was monitored. During the first few days of life the profiles were simple, but they became more complex as the bacterial diversity increased with time in both babies. Clone libraries of amplified 16S rDNA fragments from baby feces were constructed, and these libraries allowed identification of the bacterial types by comparative DNA sequence analysis; the bacteria identified included members of the generaBifidobacterium,Ruminococcus,Enterococcus,Clostridium, andEnterobacter. Species most closely related to the generaBifidobacteriumandRuminococcusin particular dominated the intestinal microbiota based on the stability over time and the numbers, as estimated by the intensities of the bands. However, 19 of the 34 cloned rDNA sequences exhibited less than 97% identity with sequences of known bacteria or cloned sequences in databases. This study showed that using PCR-DGGE and 16S rDNA sequence analysis together resulted in a dynamic description of bacterial colonization in the infant intestinal ecosystem and allowed visualization of bacteria that are difficult to cultivate or to detect by other methods.
Functional Genomics Approach to Identifying Genes Required for Biofilm Development by
Streptococcus mutans
Authors: Wen Zezhang T. , Burne Robert A. 
Published: July 2002

ABSTRACT

Streptococcus mutans
, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In
S. mutans
UA159, the
brpA
gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of
brpA
formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in
S. mutans
UA159, but insertional inactivation of the gene (
luxS
Sm
) did not alter colony or cell morphology or diminish the capacity of
S. mutans
to form biofilms. We also examined the role of the homologue of the
Bacillus subtilis
catabolite control protein CcpA in
S. mutans
in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by
S. mutans
.