bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microbial Characterization of Biofilms in Domestic Drains and the Establishment of Stable Biofilm Microcosms
Authors: McBain Andrew J. , Bartolo Robert G. , Catrenich Carl E. , Charbonneau Duane , Ledder Ruth G. , Rickard Alexander H. , Symmons Sharon A. , Gilbert Peter 
Published: January 2003

ABSTRACT

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log
10
cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log
10
cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan
Colpoda maupasi
, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.
Bacterial Competition for Human Nasal Cavity Colonization: Role of Staphylococcal
agr
Alleles
Authors: Lina Gerard , Boutite Florent , Tristan Anne , Bes Michèle , Etienne Jerome , Vandenesch Francois 
Published: January 2003

ABSTRACT

We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal
agr
alleles. The
Staphylococcus aureus
colonization rate correlated negatively with the rate of colonization by
Corynebacterium
spp. and non-
aureus
staphylococci, especially
S. epidermidis
, suggesting that both
Corynebacterium
spp. and
S. epidermidis
antagonize
S. aureus
colonization. Most of the
S. aureus
and
S. epidermidis
isolates were
agr
typed by a PCR method. Only one
S. aureus agr
(

agr
Sa

) allele was detected in each carrier. Multiple logistic regression of the two most prevalent

agr
Sa

alleles (

agr-1
Sa

and

agr-2
Sa

) and the three
S. epidermidis agr
(

agr
Se

) alleles showed a specific influence of the
agr
system. The results of this model did not support conclusions drawn from previous in vitro
agr
-specific cross-inhibition experiments. Our findings suggest that the
agr
alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties—including mediators of bacterial interference—in the strains that bear them.
Degradation of Anthracene by
Mycobacterium
sp. Strain LB501T Proceeds via a Novel Pathway, through
o
-Phthalic Acid
Authors: van Herwijnen René , Springael Dirk , Slot Pieter , Govers Harrie A. J. , Parsons John R. 
Published: January 2003

ABSTRACT

Mycobacterium
sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of
o
-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through
o
-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in
Mycobacterium
sp. LB501T.
Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene
Authors: Mancini Silvia A. , Ulrich Ania C. , Lacrampe-Couloume Georges , Sleep Brent , Edwards Elizabeth A. , Lollar Barbara Sherwood 
Published: January 2003

ABSTRACT
Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (ε) for carbon (range of −1.9 to −3.6‰) and hydrogen (range of −29 to −79‰) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.
Differential Growth Response of Colony-Forming α- and γ-Proteobacteria in Dilution Culture and Nutrient Addition Experiments from Lake Kinneret (Israel), the Eastern Mediterranean Sea, and the Gulf of Eilat
Authors: Pinhassi Jarone , Berman Tom 
Published: January 2003

ABSTRACT

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of α-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the
Roseobacter
clade, while lake dilution cultures were dominated by bacteria affiliated with the genera
Sphingomonas
and
Caulobacter
. In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing γ-proteobacteria dominated; in the marine experiments primarily different
Vibrio
and
Alteromonas
species appeared, while in the lake water experiments species of the genera
Shewanella
,
Aeromonas
, and
Rheinheimera
grew. These results suggest that major, but different, γ-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by α-proteobacteria in the presence of low nutrient concentrations.
l
-Proline Accumulation and Freeze Tolerance in
Saccharomyces cerevisiae
Are Caused by a Mutation in the
PRO1
Gene Encoding γ-Glutamyl Kinase
Authors: Morita Yuko , Nakamori Shigeru , Takagi Hiroshi 
Published: January 2003

ABSTRACT

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular
l
-proline derived from
l
-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and
l
-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-
l
-proline-utilizing strain, the mutant was found to carry an allele of the wild-type
PRO1
gene encoding γ-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of
PRO1
was shown to enhance the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of
l
-proline synthesis from
l
-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated γ-glutamyl kinase were found to accumulate intracellular
l
-proline and showed a prominent increase in cell viability after freezing at −20°C compared to the viability of cells harboring the wild-type
PRO1
gene. These results suggest that the altered γ-glutamyl kinase results in stabilization of the complex or has an indirect effect on γ-glutamyl phosphate reductase activity, which leads to an increase in
l
-proline production in
Saccharomyces cerevisiae
. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.
Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis
Authors: Temmerman R. , Scheirlinck I. , Huys G. , Swings J. 
Published: January 2003

ABSTRACT
In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.
Purification and Characterization of a Prolyl Aminopeptidase from
Debaryomyces hansenii
Authors: Bolumar Tomás , Sanz Yolanda , Aristoy M.-Concepción , Toldrá Fidel 
Published: January 2003

ABSTRACT

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of
Debaryomyces hansenii
CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate,
p
-chloromercuribenzoic acid, and Hg
2+
, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The

K
m

for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about
D. hansenii
proteases and their possible roles in meat fermentation.
Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered
Escherichia coli
without Loss of Specific Isotopic Enrichment
Authors: Sampaio Maria-Manuel , Santos Helena , Boos Winfried 
Published: January 2003

ABSTRACT

We report the construction of an
Escherichia coli
mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-
O
-α-
d
-mannosyl-
d
-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the
manA
gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the
cps
gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (
cpsG
) and mannose-1-phosphate guanylyltransferase (
cpsB
) ensure the formation of GDP-mannose. A second plasmid harbors
msg
, a gene from
Rhodothermus marinus
that encodes mannosylglycerate synthase, which catalyzes the formation of 2-
O
-α-
d
-mannosyl-
d
-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-
O
-α-
d
-mannosyl-
d
-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-α-
d
-mannosyl-
d
-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.
Induction of Sucrose Utilization Genes from
Bifidobacterium lactis
by Sucrose and Raffinose
Authors: Trindade Marla I. , Abratt Valerie R. , Reid Sharon J. 
Published: January 2003

ABSTRACT

The probiotic organism
Bifidobacterium lactis
was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of
B. lactis
was identified by complementation of a gene library in
Escherichia coli
. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The
scrP
gene was expressed constitutively from its own promoter in
E. coli
grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with
scrP
performed by using RNA isolated from
B. lactis
identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In
B. lactis
, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the
scrP
,
scrR
, and
scrT
genes in
B. lactis
indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous
E. coli
indicated that ScrR functions as a positive regulator.
Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge:
13
C Labeling and In Vivo Nuclear Magnetic Resonance
Authors: Lemos Paulo C. , Serafim Luísa S. , Santos Margarida M. , Reis Maria A. M. , Santos Helena 
Published: January 2003

ABSTRACT

In vivo
13
C and
31
P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-
13
C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV
5
, 59%; HV
4
, 5.0%; HV
3
, 1.1%; HV
2
, 3.5%; and HV
1
, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV
4
is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.
Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins
Authors: Worthington Penny , Blum Paul , Perez-Pomares Francisco , Elthon Tom 
Published: January 2003

ABSTRACT

An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon
Sulfolobus solfataricus
and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the
S
.
solfataricus
-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.
Attachment of
Listeria monocytogenes
to Radish Tissue Is Dependent upon Temperature and Flagellar Motility
Authors: Gorski Lisa , Palumbo Jeffrey D. , Mandrell Robert E. 
Published: January 2003

ABSTRACT

Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by
Listeria monocytogenes
. In order to begin to understand the physiology of the organism in a produce habitat, the ability of
L
.
monocytogenes
to attach to freshly cut radish tissue was examined. All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices. A screen was developed to identify Tn
917
-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized. Two of the three mutations were in genes with unknown functions. Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect. The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system. All three mutants were defective in attachment when tested at 30°C; the motility mutant had the most severe phenotype. However, not all of the mutants were defective when tested at other temperatures. These results indicate that
L
.
monocytogenes
may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of
Listeria
fitness in complex environments.
Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of
Cryptosporidium
Oocysts and
Giardia
Cysts in Water
Authors: McCuin Randi M. , Clancy Jennifer L. 
Published: January 2003

ABSTRACT

Collaborative and in-house laboratory trials were conducted to evaluate
Cryptosporidium
oocyst and
Giardia
cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of
Cryptosporidium
oocysts and
Giardia
cysts. Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for
Cryptosporidium
oocysts and
Giardia
cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.