bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Novel Spirochete Associated with the Hydrothermal Vent Polychaete Annelid,
Alvinella pompejana
Authors: Campbell Barbara J. , Cary S. Craig 
Published: July 2002

ABSTRACT

A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of
Alvinella pompejana
, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete
Olavius loisae
. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one
A. pompejana
specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of the
A. pompejana
specimens sampled throughout the geographic range of the worm (13°N to 32°S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of
A. pompejana
specimens tested, suggesting that a single spirochete species was present in the
A. pompejana
epibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.
Infection of
Acanthamoeba polyphaga
with
Simkania negevensis
and
S. negevensis
Survival within Amoebal Cysts
Authors: Kahane Simona , Dvoskin Bella , Mathias Mazit , Friedman Maureen G. 
Published: July 2002

ABSTRACT

Simkania negevensis
, a novel microorganism belonging to the family
Simkaniaceae
in the order
Chlamydiales
, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and
S. negevensis
has been associated with respiratory illness in humans. This study reports for the first time the ability of
S. negevensis
to survive and grow inside
Acanthamoeba polyphaga
in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of
S. negevensis
and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of
S. negevensis
in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of
S. negevensis
were observed within inclusion bodies inside
A. polyphaga
. When
S. negevensis
-infected
A. polyphaga
amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and
S. negevensis
; cysts in which
S. negevensis
cells were relegated to the space between cyst walls; and cysts containing
S. negevensis
, but apparently lacking amoebal cytoplasm.
S. negevensis
within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of
S. negevensis
needs to be investigated.
Evaluation of Two Direct Plating Methods Using Nonradioactive Probes for Enumeration of
Vibrio parahaemolyticus
in Oysters
Authors: Gooch J. A. , DePaola A. , Kaysner C. A. , Marshall D. L. 
Published: July 2002

ABSTRACT

Oysters (
Crassostrea virginica
) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine
Vibrio parahaemolyticus
densities at zero time and after 5, 10, and 24 h of postharvest storage at 26°C. After 24 h of storage at 26°C, oysters were transferred to a refrigerator at 3°C and then analyzed 14 to 17 days later. The
V. parahaemolyticus
numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (
tlh
), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (
r
= 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (
r
= 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total
V. parahaemolyticus
, and they were more rapid and less labor-intensive.
Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from
Rhodococcus erythropolis
DCL14
Authors: van der Vlugt-Bergmans Cécile J. B , van der Werf Mariët J. 
Published: July 2002

ABSTRACT

A monoterpene ɛ-lactone hydrolase (MLH) from
Rhodococcus erythropolis
DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4
R
)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6
R
)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4
R
)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated
mlhB
(monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.
Protein
trans
-Splicing To Produce Herbicide-Resistant Acetolactate Synthase
Authors: Sun Luo , Ghosh Inca , Paulus Henry , Xu Ming-Qun 
Published: July 2002

ABSTRACT

Protein splicing in
trans
has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by
trans
-splicing has not been reported previously. In this study, we used the DnaE intein from
Synechocystis
sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by
trans
-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in
Escherichia coli
. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in
trans
and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of
E. coli
with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host
E. coli
cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.
Predictive Modeling of the Shelf Life of Fish under Nonisothermal Conditions
Authors: Koutsoumanis Konstantinos 
Published: July 2002

ABSTRACT

The behavior of the natural microflora of Mediterannean gilt-head seabream (
Sparus aurata
) was monitored during aerobic storage at different isothermal conditions from 0 to 15°C. The growth data of pseudomonads, established as the specific spoilage organisms of aerobically stored gilt-head seabream, combined with data from previously published experiments, were used to model the effect of temperature on pseudomonad growth using a Belehradek type model. The nominal minimum temperature parameters of the Belehradek model (
T
min
) for the maximum specific growth rate (μ
max
) and the lag phase (
t
Lag
) were determined to be −11.8 and −12.8°C, respectively. The applicability of the model in predicting pseudomonad growth on fish at fluctuating temperatures was evaluated by comparing predictions with observed growth in experiments under dynamic conditions. Temperature scenarios designed in the laboratory and simulation of real temperature profiles observed in the fish chill chain were used. Bias and accuracy factors were used as comparison indices and ranged from 0.91 to 1.17 and from 1.11 to 1.17, respectively. The average percent difference between shelf life predicted based on pseudomonad growth and shelf life experimentally determined by sensory analysis for all temperature profiles tested was 5.8%, indicating that the model is able to predict accurately fish quality in real-world conditions.
Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR
Authors: Tajima K. , Aminov R. I. , Nagamine T. , Matsui H. , Nakamura M. , Benno Y. 
Published: July 2002

ABSTRACT

A set of PCR primers was designed and validated for specific detection and quantification of
Prevotella ruminicola
,
Prevotella albensis
,
Prevotella bryantii
,
Fibrobacter succinogenes
,
Selenomonas ruminantium-Mitsuokella multiacida
,
Streptococcus bovis
,
Ruminococcus flavefaciens
,
Ruminobacter amylophilus
,
Eubacterium ruminantium
,
Treponema bryantii
,
Succinivibrio dextrinosolvens
, and
Anaerovibrio lipolytica
. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria,
F. succinogenes
and
R. flavefaciens
, were in agreement with those of earlier, culture-based experiments. The quantity of
F. succinogenes
DNA
,
predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The
R. flavefaciens
DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of
P. ruminicola
increased 7-fold and that of
P. bryantii
increased 263-fold. On day 28, the quantity of
P. ruminicola
DNA decreased 3-fold, while
P. bryantii
DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium,
E. ruminantium,
dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete,
T. bryantii,
decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in
A. lipolytica
DNA were not statistically significant. After an initial slight increase in
S. dextrinosolvens
DNA on day 3, this DNA was not detected at the end of the experiment.
S. bovis
DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of
S. ruminantium
-
M. multiacida
DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.
Identification and Functional Characterization of CbaR, a MarR-Like Modulator of the
cbaABC
-Encoded Chlorobenzoate Catabolism Pathway
Authors: Providenti Miguel A. , Wyndham R. Campbell 
Published: July 2002

ABSTRACT

In
Comamonas testosteroni
BR60 (formerly
Alcaligenes
sp. strain BR60), catabolism of the pollutant 3-chlorobenzoate (3CBA) is initiated by enzymes encoded by
cbaABC
, an operon found on composite transposon Tn
5271
of plasmid pBRC60. The
cbaABC
gene product CbaABC converts 3CBA to protocatechuate (PCA) and 5-Cl-PCA, which are then metabolized by the chromosomal PCA
meta
(extradiol) ring fission pathway. In this study,
cbaA
was found to possess a ς
70
type promoter. O
2
uptake experiments with whole cells and expression studies with
cbaA
-
lacZ
constructs showed that
cbaABC
was induced by 3CBA. Benzoate, which is not a substrate of the 3CBA pathway, was a gratuitous inducer, and CbaR, a MarR family repressor coded for by a divergently transcribed gene upstream of
cbaABC
, could modulate induction mediated by benzoate. Purified CbaR bound specifically to two regions of the
cbaA
promoter (P

cbaA

); site I, a high-affinity site, is between the transcriptional start point (position +1) and the start codon of
cbaA
, while site II, a lower-affinity site, overlaps position +1. 3CBA at concentrations as low as 40 μM interfered with binding to P

cbaA

. PCA also interfered with binding, while benzoate only weakly disrupted binding. Unexpectedly, benzoate with a hydroxyl or carboxyl at position 3 improved CbaR binding. Data are also presented that suggest that an unidentified regulator is encoded on the chromosome that induces
cbaABC
in response to benzoate and 3CBA.
Characterization ofSchizosaccharomyces pombeMalate Permease by Expression inSaccharomyces cerevisiae
Authors: Camarasa Carole , Bidard Frédérique , Bony Muriel , Barre Pierre , Dequin Sylvie 
Published: July 2002

ABSTRACTInSaccharomyces cerevisiae,l-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression inS. cerevisiaeof theMAE1gene, encodingSchizosaccharomyces pombemalate permease, markedly increasedl-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes),l-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters:Vmax= 8.7 nmol/mg/min;Km= 1.6 mM) and some simple diffusion of the undissociatedl-malic acid (Kd= 0.057 min−1). As totall-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain.l-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the ΔpH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related tol-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibitedl-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. InS. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediatedl-malic acid import inS. cerevisiae, and intracellular pH remained constant. This suggests that, inS. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.
Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in
Aspergillus parasiticus
Authors: Chiou Ching-Hsun , Miller Michael , Wilson David L. , Trail Frances , Linz John E. 
Published: July 2002

ABSTRACT

The
nor-1
gene in the filamentous fungus
Aspergillus parasiticus
encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on
nor-1
expression, we generated plasmid pAPGUSNNB containing a
nor-1
promoter-β-glucuronidase (GUS) (encoded by
uidA
) reporter fusion with
niaD
(encodes nitrate reductase) as a selectable marker.
niaD
transformants of
A. parasiticus
strain NR-1 (
niaD
) carried pAPGUSNNB integrated predominantly at the
nor-1
or
niaD
locus. Expression of the native
nor-1
and
nor-1
::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of
nor-1
promoter function with pAPGUSNNB integrated at
nor-1
was similar to that observed for the native
nor-1
gene. In contrast,
nor-1
promoter activity in pAPGUSNNB and a second
nor-1
::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at
niaD
. Because
niaD
-dependent regulation could account for the absence of expression at
niaD
, a third chromosomal location was analyzed using pAPGUSNP, which contained
nor-1
::GUS plus
pyrG
(encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at
nor-1
and was not detectable at
pyrG
, even under growth conditions that required
pyrG
expression.
nor-1
::GUS is regulated similarly to the native
nor-1
gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native
nor-1
levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure
nor-1
promoter activity and that
nor-1
is subject to position-dependent regulation in the
A. parasiticus
chromosome.
Isogenic Strain of
Escherichia coli
O157:H7 That Has Lost both Shiga Toxin 1 and 2 Genes
Authors: Feng Peter , Dey Manashi , Abe Akio , Takeda Tae 
Published: July 2002

ABSTRACT

An
Escherichia coli
O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit β-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93
gusA
mutation, the cluster I
ehxA
gene for enterohemolysin, and the
eae
gene for γ-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of
Xba
I-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both
stx
genes.
Use of Recombinant BP26 Protein in Serological Diagnosis of
Brucella melitensis
Infection in Sheep
Authors: Cloeckaert Axel , Baucheron Sylvie , Vizcaino Nieves , Zygmunt Michel S. 
Published: July 2002

ABSTRACT

Previously a
Brucella
protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and
B. melitensis
Rev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in
Escherichia coli
in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (
n
= 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (
n
= 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (
n
= 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (
n
= 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with
B. melitensis
H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and
B. melitensis
Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that a
B. melitensis
Rev.1
bp26
gene deletion mutant should be constructed to ensure this differentiation.
Toxin Gene Analysis of a Variant Strain of
Clostridium difficile
That Causes Human Clinical Disease
Authors: Sambol Susan P. , Merrigan Michelle M. , Lyerly David , Gerding Dale N. , Johnson Stuart 
Published: July 2002

ABSTRACT

A toxin variant strain of
Clostridium difficile
was isolated from two patients with
C. difficile
-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for
C. difficile
toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (
tcdA
) and toxin B (
tcdB
) genes of type CF2 isolate 5340. Type CF2 5340
tcdA
exhibited a 1,821-bp truncation, due to three deletions in the 3′ end of the gene, and a point mutation in the 5′ end of the gene, resulting in a premature stop codon at
tcdA
position 139. Type CF2 5340
tcdB
exhibited multiple nucleotide base substitutions in the 5′ end of the gene compared to
tcdB
of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant
C. difficile
strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3′ end of
tcdA
demonstrated identical 1.8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of
C. difficile
that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.