bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variable Bacteriocin Production in the Commercial Starter
Lactococcus lactis
DPC4275 Is Linked to the Formation of the Cointegrate Plasmid pMRC02
Authors: Trotter Maeve , McAuliffe Olivia E. , Fitzgerald Gerald F. , Hill Colin , Ross R. Paul , Coffey Aidan 
Published: January 2004

ABSTRACT

Lactococcus lactis
DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with
L. lactis
MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage
c
2 infection of MG1363(pMRC02). While this locus is flanked by IS
946
elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.
Degradation of Benzo[
a
]pyrene by
Mycobacterium vanbaalenii
PYR-1
Authors: Moody Joanna D. , Freeman James P. , Fu Peter P. , Cerniglia Carl E. 
Published: January 2004

ABSTRACT

Metabolism of the environmental pollutant benzo[
a
]pyrene in the bacterium
Mycobacterium vanbaalenii
PYR-1 was examined. This organism initially oxidized benzo[
a
]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[
a
]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were
cis
-4,5-dihydro-4,5-dihydroxybenzo[
a
]pyrene (benzo[
a
]pyrene
cis
-4,5-dihydrodiol),
cis
-11,12-dihydro-11,12-dihydroxybenzo[
a
]pyrene (benzo[
a
]pyrene
cis
-11,12-dihydrodiol),
trans
-11,12-dihydro-11,12-dihydroxybenzo[
a
]pyrene (benzo[
a
]pyrene
trans
-11,12-dihydrodiol), 10-oxabenzo[
def
]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[
a
]pyrene. The
ortho
-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[
a
]pyrene
cis
-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[
a
]pyrene
cis
-4,5-dihydrodiol had 30% 4
S
,5
R
and 70% 4
R
,5
S
absolute stereochemistry. Benzo[
a
]pyrene
cis
-11,12-dihydrodiol adopted an 11
S
,12
R
conformation with 100% optical purity. The enantiomeric composition of benzo[
a
]pyrene
trans
-11,12-dihydrodiol was an equal mixture of 11
S
,12
S
and 11
R
,12
R
molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[
a
]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[
a
]pyrene in
M. vanbaalenii
PYR-1.
Frequency, Size, and Localization of Bacterial Aggregates on Bean Leaf Surfaces
Authors: Monier J.-M. , Lindow S. E. 
Published: January 2004

ABSTRACT

Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium
Pseudomonas syringae
pv. syringae strain B728a. Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10
4
cells per aggregate. The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions. The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution. While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present. We observed that up to 50% of cells present on a leaf were located in aggregates containing 10
3
cells or more. Aggregates were associated with several different anatomical features of the leaf surface but not with stomates. Aggregates were preferentially associated with glandular trichomes and veins. The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed.
Occurrence of Genes Associated with Enterotoxigenic and Enterohemorrhagic
Escherichia coli
in Agricultural Waste Lagoons
Authors: Chern Eunice C. , Tsai Yu-Li , Olson Betty H. 
Published: January 2004

ABSTRACT

The prevalence among all
Escherichia coli
bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic
E. coli
, and of three genes (
stxI
,
stxII
, and
eaeA
) associated with enterohemorrhagic
E. coli
was determined in farm waste disposal systems seasonally for 1 year. Single- and nested-PCR results for the number of
E. coli
isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method. The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested. Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the
stxI
,
stxII
, and
eaeA
genes. The geometric mean prevalence of each toxin gene within the
E. coli
community in waste disposal site waters after nested MPN-PCR was 1:8.5
E. coli
isolates (1:8.5
E. coli
) for the LTIIa toxin gene and 1:4
E. coli
for the STII toxin gene. The geometric mean prevalence for the simultaneous occurrence of toxin genes
stxI
,
stxII
, and
eaeA
, was 1:182
E. coli
. These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates. With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans.
Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by
Mycobacterium
sp. Strain RJGII-135
Authors: Miyata Naoyuki , Iwahori Keisuke , Foght Julia M. , Gray Murray R. 
Published: January 2004

ABSTRACT

The mechanism of uptake of phenanthrene by
Mycobacterium
sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-
14
C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state
14
C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide
m
-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (

K
t

) of 26 ± 3 nM (mean ± standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated
14
C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (

K
d

) of 41 ± 21 nM (mean ± standard deviation). Given the low values of

K
t

and

K
d

,
Mycobacterium
sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.
Overproduction of Trehalose: Heterologous Expression of
Escherichia coli
Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in
Corynebacterium glutamicum
Authors: Padilla Leandro , Krämer Reinhard , Stephanopoulos Gregory , Agosin Eduardo 
Published: January 2004

ABSTRACT

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of
Corynebacterium glutamicum
. This paper describes the heterologous expression of
Escherichia coli
trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in
C. glutamicum
, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in
C. glutamicum
to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in
C. glutamicum
.
Longitudinal Study of Fecal Shedding of
Escherichia coli
O157:H7 in Feedlot Cattle: Predominance and Persistence of Specific Clonal Types despite Massive Cattle Population Turnover
Authors: LeJeune J. T. , Besser T. E. , Rice D. H. , Berg J. L. , Stilborn R. P. , Hancock D. D. 
Published: January 2004

ABSTRACT

Identification of the sources and methods of transmission of
Escherichia coli
O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of
E. coli
O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation,
E. coli
O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of
E. coli
O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of
Xba
I-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of
E. coli
O157:H7 on farms.
Microbial Anaerobic Demethylation and Dechlorination of Chlorinated Hydroquinone Metabolites Synthesized by Basidiomycete Fungi
Authors: Milliken C. E. , Meier G. P. , Watts J. E. M. , Sowers K. R. , May H. D. 
Published: January 2004

ABSTRACTThe synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed thatDesulfitobacterium hafniensestrains DCB2 and PCP1,Desulfitobacterium chlororespiransstrain Co23, andDesulfitobacterium dehalogenansJW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.
Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific
Authors: Nakagawa Tatsunori , Ishibashi Jun-Ichiro , Maruyama Akihiko , Yamanaka Toshiro , Morimoto Yusuke , Kimura Hiroyuki , Urabe Tetsuro , Fukui Manabu 
Published: January 2004

ABSTRACTThis study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with theAquificaeand γ- and ε-Proteobacteriaas well as the occurrence of archaeal phylotypes affiliated with theThermococcalesand of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genusThiomicrospira, andPelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.
Growth of
Escherichia coli
Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source
Authors: McLoughlin Sean Yu , Jackson Colin , Liu Jian-Wei , Ollis David L. 
Published: January 2004

ABSTRACT

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The
Escherichia coli
strain DH10B expressing the phosphotriesterase from
Agrobacterium radiobacter
P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source.
Enterobacter aerogenes
is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled
E. coli
to grow when dimethyl phosphate is the sole phosphorus source.
E. coli
coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled
E. coli
to use phosphotriesters as a sole source of phosphorus.
Periplasmic Cytochrome
c
3
of
Desulfovibrio vulgaris
Is Directly Involved in H
2
-Mediated Metal but Not Sulfate Reduction
Authors: Elias Dwayne A. , Suflita Joseph M. , McInerney Michael J. , Krumholz Lee R. 
Published: January 2004

ABSTRACT

Kinetic parameters and the role of cytochrome
c
3
in sulfate, Fe(III), and U(VI) reduction were investigated in
Desulfovibrio vulgaris
Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (

K
m

= 220 μM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H
2
and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H
2
and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine
c
-type cytochrome involvement in electron acceptor reduction.
c
-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H
2
, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the
c
-type cytochrome in lactate- or pyruvate-reduced, but not H
2
-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H
2
was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H
2
was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome
c
3
is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.
Supplementation of
Perkinsus marinus
Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity
Authors: Earnhart Christopher G. , Vogelbein Mary Ann , Brown Gwynne D. , Reece Kimberly S. , Kaattari Stephen L. 
Published: January 2004

ABSTRACT

The protozoan oyster parasite
Perkinsus marinus
can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of
P. marinus
-susceptible (
Crassostrea virginica
) and -tolerant (
Crassostrea gigas
,
Crassostrea ariakensis
) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.