bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of an Inducible Chlorophenol
O
-Methyltransferase from
Trichoderma longibrachiatum
Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines
Authors: Coque Juan-José R. , Álvarez-Rodríguez María Luisa , Larriba Germán 
Published: September 2003

ABSTRACT

A novel
S
-adenosyl-
l
-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of
Trichoderma longibrachiatum
CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [
methyl
-
3
H]SAM, and this reaction was prevented by previous incubation with
S
-adenosylhomocysteine. Gel filtration indicated that the
M
r
was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The

K
m

values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 μM, respectively.
S
-Adenosylhomocysteine acted as a competitive inhibitor, with a

K
i

of 378.9 ± 45.4 μM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu
2+
, Hg
2+
, Zn
2+
, and Ag
+
, and to a lesser extent by
p
-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.
Purification and Functional Characterization of a Novel α-
l
-Arabinofuranosidase from
Bifidobacterium longum
B667
Authors: Margolles Abelardo , de los Reyes-Gavilán Clara G. 
Published: September 2003

ABSTRACT

The gene encoding a novel α-
l
-arabinofuranosidase from
Bifidobacterium longum
B667,
abfB
, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-
l
-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in
Lactococcus lactis
under control of the tightly regulated, nisin-inducible
nisA
promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70°C), and it had an optimum pH of 6.0 and an optimum temperature of 45°C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55°C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu
2+
, Hg
2+
, and Zn
2+
markedly reduced enzymatic activity. The protein exhibited a high level of activity with
p
-nitrophenyl α-
l
-arabinofuranoside, with apparent
K

m

and
V
max
values of 0.295 mM and 417 U/mg, respectively. AbfB released
l
-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of
l
-arabinose-containing polysaccharides.
Sequence Diversity and Functional Conservation of the Origin of Replication in Lactococcal Prolate Phages
Authors: Rakonjac Jasna , Ward Lawrence J. H. , Schiemann Anja H. , Gardner Paul P. , Lubbers Mark W. , O'Toole Paul W. 
Published: September 2003

ABSTRACT

Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium
Lactococcus lactis
. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P
E
1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P
E
1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P
E
1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.
Heat Adaptation Alters
Escherichia coli
O157:H7 Membrane Lipid Composition and Verotoxin Production
Authors: Yuk Hyun-Gyun , Marshall Douglas L. 
Published: September 2003

ABSTRACT

The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and verotoxin concentration of
Escherichia coli
O157:H7 (ATCC 43895), an
rpoS
mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant
E. coli
O157:H7 (B6-914), and nonpathogenic
E. coli
(ATCC 25922) was investigated.
D
values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and
cis
-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the
rpoS
mutant, whereas there was a reduction and no change in the amount of
cis
-vaccenic acid in nonpathogenic and verotoxin mutant
E. coli
, respectively. The ratio of palmitic to
cis
-vaccenic acids decreased in ATCC 43895 and in the
rpoS
mutant, whereas the ratio increased in nonpathogenic
E. coli
and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and
rpoS
mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The
rpoS
gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.
Genetic Engineering of a Highly Solvent-Tolerant
Pseudomonas putida
Strain for Biotransformation of Toluene to
p-
Hydroxybenzoate
Authors: Ramos-González María-Isabel , Ben-Bassat Arie , Campos María-Jesús , Ramos Juan L. 
Published: September 2003

ABSTRACT

The solvent-tolerant strain
Pseudomonas putida
DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA).
P. putida
DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase. The
todC1C2
genes encode the α and β subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1EΔ
todC
mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The
P. putida pobA
gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a
Pseudomonas mendocina pobA1 pobA2
double mutant. This
pobA
gene was knocked out in vitro and used to generate a double mutant, DOT-T1EΔ
todCpobA
, that was unable to use either toluene or 4-HBA as a carbon source. The
tmo
and
pcu
genes from
P. mendocina
KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of
p-
cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn
5Tc
and stably recruited in the chromosome of DOT-T1EΔ
todCpobA
. Expression of the
tmo
and
pcu
genes took place in a DOT-T1E background due to cross-activation of the
tmo
promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.
Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of
Bacillus
and Related Genera
Authors: Daffonchio Daniele , Cherif Ameur , Brusetti Lorenzo , Rizzi Aurora , Mora Diego , Boudabous Abdellatif , Borin Sara 
Published: September 2003

ABSTRACT

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera
Bacillus
,
Brevibacillus
,
Geobacillus
, and
Paenibacillus
. We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.
Predictive Thermal Inactivation Model for Effects of Temperature, Sodium Lactate, NaCl, and Sodium Pyrophosphate on
Salmonella
Serotypes in Ground Beef
Authors: Juneja Vijay K. , Marks Harry M. , Mohr Tim 
Published: September 2003

ABSTRACT

Analyses
of survival data of a mixture of
Salmonella
spp. at fixed
temperatures between 55°C (131°F) and 71.1°C
(160°F) in ground beef matrices containing concentrations of
salt between 0 and 4.5%, concentrations of sodium pyrophosphate
(SPP) between 0 and 0.5%, and concentrations of sodium lactate
(NaL) between 0 and 4.5% indicated that heat resistance of
Salmonella
increases with increasing levels of SPP and salt,
except that, for salt, for larger lethalities close to 6.5, the effect
of salt was evident only at low temperatures (<64°C).
NaL did not seem to affect the heat resistance of
Salmonella
as much as the effects induced by the other variables studied. An
omnibus model for predicting the lethality for given times and
temperatures for ground beef matrices within the range studied was
developed that reflects the convex survival curves that were observed.
However, the standard errors of the predicted lethalities from this
models are large, so consequently, a model, specific for predicting the
times needed to obtained a lethality of 6.5 log
10
, was
developed, using estimated results of times derived from the individual
survival curves. For the latter model, the coefficient of variation
(CV) of predicted times range from about 6 to 25%. For example,
at 60°C, when increasing the concentration of salt from 0 to
4.5%, and assuming that the concentration of SPP is 0%,
the time to reach a 6.5-log
10
relative reduction is
predicted to increase from 20 min (CV = 11%) to 48 min
(CV = 15%), a 2.4 factor (CV = 19%). At
71.1°C (160°F) the model predicts that more than 0.5
min is needed to achieve a 6.5-log
10
relative
reduction.
Cyanobacterial Diversity in Natural and Artificial Microbial Mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a Morphological and Molecular Approach
Authors: Taton Arnaud , Grubisic Stana , Brambilla Evelyne , De Wit Rutger , Wilmotte Annick 
Published: September 2003

ABSTRACT

Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from
Nodularia
and
Hydrocoryne
, as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.
Impact of Seasonal Variations and Nutrient Inputs on Nitrogen Cycling and Degradation of Hexadecane by Replicated River Biofilms
Authors: Chénier Martin R. , Beaumier Danielle , Roy Réal , Driscoll Brian T. , Lawrence John R. , Greer Charles W. 
Published: September 2003

ABSTRACT

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both
alkB
and
nirK
were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for
nirK
from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.
Real-Time PCR for Quantification of
Giardia
and
Cryptosporidium
in Environmental Water Samples and Sewage
Authors: Guy Rebecca A. , Payment Pierre , Krull Ulrich J. , Horgen Paul A. 
Published: September 2003

ABSTRACT

The protozoan pathogens
Giardia lamblia
and
Cryptosporidium parvum
are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of
Giardia
and
Cryptosporidium
are very low. Primers and TaqMan probes based on the β-giardin gene of
G. lamblia
and the COWP gene of
C. parvum
were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of
G. lamblia
and one oocyst of
C. parvum
. A multiplex real-time PCR (qPCR) assay for simultaneous detection of
G. lamblia
and
C. parvum
resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify
G. lamblia
in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of
G. lamblia
in the sewage. No
Cryptosporidium
was detected in these samples by either method.
Biogeography of the Purple Nonsulfur Bacterium
Rhodopseudomonas palustris
Authors: Oda Yasuhiro , Star Bastiaan , Huisman Louis A. , Gottschal Jan C. , Forney Larry J. 
Published: September 2003

ABSTRACT

The biogeography of the purple nonsulfur bacterium
Rhodopseudomonas palustris
on a local scale was investigated. Thirty clones of phototrophic bacteria were isolated from each of five unevenly spaced sampling locations in freshwater marsh sediments along a linear 10-m transect, and a total of 150 clones were characterized by BOX-PCR genomic DNA fingerprinting. Cluster analysis of 150 genomic fingerprints yielded 26 distinct genotypes, and 106 clones constituted four major genotypes that were repeatedly isolated. Representatives of these four major genotypes were tentatively identified as
R. palustris
based on phylogentic analyses of 16S rRNA gene sequences. The differences in the genomic fingerprint patterns among the four major genotypes were accompanied by differences in phenotypic characteristics. These phenotypic differences included differences in the kinetics of carbon source use, suggesting that there may be functional differences with possible ecological significance among these clonal linages. Morisita-Horn similarity coefficients (
C
MH
), which were used to compare the numbers of common genotypes found at pairs of sampling locations, showed that there was substantial similarity between locations that were 1 cm apart (
C
MH
, ≥0.95) but there was almost no similarity between locations that were ≥9 m apart (
C
MH
, ≤0.25). These calculations showed there was a gradual decrease in similarity among the five locations as a function of distance and that clones of
R. palustris
were lognormally distributed along the linear 10-m transect. These data indicate that natural populations of
R. palustris
are assemblages of genetically distinct ecotypes and that the distribution of each ecotype is patchy.
Spatial Distribution of
Rhodopseudomonas palustris
Ecotypes on a Local Scale
Authors: Bent S. J. , Gucker C. L. , Oda Y. , Forney L. J. 
Published: September 2003

ABSTRACT

The number, spatial distribution, and significance of genetically distinguishable ecotypes of prokaryotes in the environment are poorly understood. Oda et al. (Y. Oda, B. Star, L. A. Huisman, J. C. Gottschal, and L. J. Forney, Appl. Environ. Microbiol. 69:xxx-xxx, 2003) have shown that
Rhodopseudomonas palustris
ecotypes were lognormally distributed along a 10-m transect and that multiple strains of the species could coexist in 0.5-g sediment samples. To extend these observations, we investigated the clonal diversity of
R. palustris
in 0.5-g samples taken from the corners and center of a 1-m square. A total of 35 or 36 clones were recovered by direct plating from each sample and were characterized by BOX A1R repetitive element-PCR genomic DNA fingerprinting. Isolates with fingerprint images that were ≥80% similar to each other were defined as the same genotype. Among the 178 isolates studied, 32 genotypes were identified, and each genotype contained between 1 and 40 isolates. These clusters were consistent with minor variations found in 16S rRNA gene sequences. The Shannon indices of the genotypic diversity within each location ranged from 1.08 (5 genotypes) to 2.18 (13 genotypes). Comparison of the rank abundance of genotypes found in pairs of locations showed that strains from three locations were similar to each other, with Morisita-Horn similarity coefficients ranging from 0.59 to 0.71. All comparisons involving the remaining two locations resulted in coefficients between 0 and 0.12. From these results we inferred that the patterns of ecotype diversity at the sampling site are patchy at a 1-m scale and postulated that factors such as mixing, competitive interactions, and microhabitat variability are likely to be responsible for the maintenance of the similarities between some locations and the differences between others.