bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Functional Characterization of IS
1358
from
Vibrio cholerae
Authors: Dumontier Sandrine , Trieu-Cuot Patrick , Berche Patrick 
Published: March 2020

ABSTRACT

The new epidemic serovar O139 of
Vibrio cholerae
has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS
1358
O139
. We studied the distribution of the IS
1358
element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS
1358
was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS
1358
from
V. cholerae
O22 and one copy from
V. cholerae
O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS
1358
to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an
Escherichia coli recA
donor at a frequency of 1.2 × 10
−8
. Sequence analysis revealed that IS
1358
duplicates 10 bp at its insertion site.
H-NS and StpA Proteins Stimulate Expression of the Maltose Regulon in
Escherichia coli
Authors: Johansson Jörgen , Dagberg Björn , Richet Evelyne , Uhlin Bernt Eric 
Published: March 2020

ABSTRACT

The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in
Escherichia coli
. Its role in gene regulation is manifested by the increased expression of several gene products in
hns
mutant strains. Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon. In studies with
hns
mutant strains and derivatives also deficient in the
stpA
gene, we found that expression of the LamB porin was decreased. Our results showed that the amounts of both LamB protein and
lamB
mRNA were greatly reduced in
hns
and
hns-stpA
mutant strains. The same results were obtained when we monitored the amount of transcription from the
malEFG
operon. The
lamB
gene is situated in the
malKlamBmalM
operon, which forms a divergent operon complex together with the
malEFG
operon. The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein. Using a
malT-lacZ
translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in
hns
and
hns-stpA
mutant strains in comparison with the wild-type strain. Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon.
Molecular Characterization of a Stress-Inducible Gene from
Lactobacillus helveticus
Authors: Smeds Andrèas , Varmanen Pekka , Palva Airi 
Published: March 2020

ABSTRACT

A gene (
htrA
) coding for a stress-inducible HtrA-like protein from
Lactobacillus helveticus
CNRZ32 was cloned, sequenced, and characterized. The deduced amino acid sequence of the gene exhibited 30% identity with the HtrA protein from
Escherichia coli
; the putative catalytic triad and a PDZ domain that characterize the HtrA family of known bacterial serine proteases were also found in the sequence. Expression of the
L. helveticus htrA
gene in a variety of stress conditions was analyzed at the transcriptional level. The strongest induction, resulting in over an eightfold increase in the
htrA
transcription level, was found in growing CNRZ32 cells exposed to 4% (wt/vol) NaCl. Enhanced
htrA
mRNA expression was also seen in CNRZ32 cells after exposure to puromycin, ethanol, or heat. The reporter gene
gusA
was integrated in the
Lactobacillus
chromosome downstream of the
htrA
promoter by a double-crossover event which also interrupted the wild-type gene. The expression of
gusA
in the stress conditions tested was similar to that of
htrA
itself. In addition, the presence of an intact
htrA
gene facilitated growth under heat stress but not under salt stress.
Catabolite Regulation of the
Bacillus subtilis ctaBCDEF
Gene Cluster
Authors: Liu Xuemin , Taber Harry W. 
Published: March 2020

ABSTRACT

Bacillus subtilis
cytochrome
c
oxidase
caa
3
is encoded by the
ctaCDEF
genes at the
ctaABCDEF
locus, with the
ctaBCDEF
genes organized as an operon-like unit. A dyad symmetry sequence and a catabolite response element homolog can be recognized in the 240-bp intercistronic region between
ctaB
and
ctaC. ctaB′-lacZ
and
ctaBCD′-lacZ
transcriptional fusions integrated at the native locus were used to study catabolite effects on transcription of the
ctaB
and
ctaCDEF
genes. In Schaeffer’s medium lacking glucose,
ctaBCD′-lacZ
was expressed at a very low level during the exponential phase, and expression increased about 30-fold 2 h after entry into the stationary phase. In the presence of 0.5% glucose,
ctaBCD′-lacZ
expression was totally repressed. In contrast to
ctaBCD′-lacZ
,
ctaB′-lacZ
was constitutively expressed regardless of carbon source. The
ctaCDEF
genes were separated from
ctaB
by insertion of plasmids carrying selectable markers in such a way that the
ctaCDEF
and
ctaB
transcription units remained intact. Enzymatic assays of
caa
3
with these constructs, showed that
ctaCDEF
was not expressed independently of
ctaB
. Also, when a ′
ctaB-ctaC′-lacZ
fusion (containing the
ctaB-ctaC
intercistronic region) was placed at a remote nonessential locus, β-galactosidase activity could not be detected. The absence of a promoter in the
ctaB-ctaC
intercistronic space also was indicated by the inability to detect
ctaC
-specific transcripts with RNase protection assays, primer extension, and rapid amplification of 5′ cDNA ends. Direct mRNA measurements showed that, in the presence of 0.5% glucose,
ctaBCDEF
transcripts terminated at the 3′ end of the putative stem-loop structure and the distal portion was down-regulated. A possible mechanism for
ctaCDEF
gene regulation is suggested. Catabolite repression of
ctaBCD′-lacZ
was partly dependent on CcpA but was independent of HPr. The expression of
ctaBCDEF
also appears to require the
strC
,
ctaA
, and
resD-resE
gene products.
Gene Cloning and Characterization of Recombinant RNase HII from a Hyperthermophilic Archaeon
Authors: Haruki Mitsuru , Hayashi Keiko , Kochi Takayuki , Muroya Ayumu , Koga Yuichi , Morikawa Masaaki , Imanaka Tadayuki , Kanaya Shigenori 
Published: March 2020

ABSTRACT

We have cloned the gene encoding RNase HII (RNase HII
Pk
) from the hyperthermophilic archaeon
Pyrococcus kodakaraensis
KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an
rnh
mutant strain of
Escherichia coli
. This gene was expressed in an
rnh
mutant strain of
E. coli
, the recombinant enzyme was purified, and its biochemical properties were compared with those of
E. coli
RNases HI and HII. RNase HII
Pk
is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of
Methanococcus jannaschii
,
Saccharomyces cerevisiae
, and
E. coli
RNase HII proteins, respectively. The enzymatic activity was determined at 30°C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co
2+
for RNase HII
Pk
, Mn
2+
for
E. coli
RNase HII, and Mg
2+
for
E. coli
RNase HI. The specific activity of RNase HII
Pk
determined in the presence of the most preferred metal ion was 6.8-fold higher than that of
E. coli
RNase HII and 4.5-fold lower than that of
E. coli
RNase HI. Like
E. coli
RNase HI, RNase HII
Pk
and
E. coli
RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3′ bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HII
Pk
and
E. coli
RNases HI and HII are structurally and functionally related to one another.
Methionine Induces Sexual Development in the Fission Yeast
Schizosaccharomyces pombe
via an
ste11
-Dependent Signalling Pathway
Authors: Schweingruber Anne-Marie , Hilti Norma , Edenharter Eleonore , Schweingruber M. Ernst 
Published: March 2020

ABSTRACT

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of
Schizosaccharomyces pombe
. In heterothallic strains it induces G
1
arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including
fbp1
and
ste11
. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent
ste11
signalling pathway.
A Family of Stability Determinants in Pathogenic Bacteria
Authors: Hayes Finbarr 
Published: March 2020

ABSTRACT

A novel segregational stability system was identified on plasmid R485, which originates from
Morganella morganii
. The system is composed of two overlapping genes,
stbD
and
stbE
, which potentially encode proteins of 83 and 93 amino acids, respectively. Homologs of the
stbDE
genes were identified on the enterotoxigenic plasmid P307 from
Escherichia coli
and on the chromosomes of
Vibrio cholerae
and
Haemophilus influenzae
biogroup
aegyptius
. The former two homologs also promote plasmid stability in
E. coli
. Furthermore, the
stbDE
genes share homology with components of the
relBEF
operon and with the
dnaT
gene of
E. coli
. The organization of the
stbDE
cassette is reminiscent of toxin-antitoxin stability cassettes.
secG
and Temperature Modulate Expression of Azide-Resistant and Signal Sequence Suppressor Phenotypes of
Escherichia coli secA
Mutants
Authors: Ramamurthy Visvanathan , Dapíc Vesna , Oliver Donald 
Published: March 2020

ABSTRACT

SecA is a dynamic protein that undergoes ATP-dependent membrane cycling to drive protein translocation across the
Escherichia coli
inner membrane. To understand more about this process, azide-resistant (
azi
) and signal sequence suppressor (
prlD
) alleles of
secA
were studied. We found that azide resistance is cold sensitive because of a direct effect on protein export, suggesting that SecA-membrane interaction is regulated by an endothermic step that is azide inhibitable.
secG
function is required for expression of azide-resistant and signal sequence suppressor activities of
azi
and
prlD
alleles, and in turn, these alleles suppress cold-sensitive and export-defective phenotypes of a
secG
null mutant. These remarkable genetic observations support biochemical data indicating that SecG promotes SecA membrane cycling and that this process is dependent on an endothermic change in SecA conformation.
Isolation in Endothelial Cell Cultures of
Chlamydia trachomatis
LGV (Serovar L2) from a Lymph Node of a Patient with Suspected Cat Scratch Disease
Authors: Maurin M. , Raoult D. 
Published: March 2020

ABSTRACT

An inguinal lymph node, removed from a 21-year-old Romanian man suspected of having cat scratch disease, was sent to our laboratory for
Bartonella
culture. Lymph node specimens were inoculated on blood-enriched agar and in an endothelial cell culture system using the centrifugation shell vial technique. Bacteria were grown in cell monolayers and detected as positive with an anti-
Bartonella henselae
rabbit serum. However, such bacteria were identified as
Chlamydia trachomatis
biovar LGV serovar L2 by PCR sequencing techniques. Pathological examination of tissue biopsies was compatible with either lymphogranuloma venereum or cat scratch disease. The shell vial system is suitable for isolation of intracellular pathogens responsible for chronic lymphadenopathies, including
C. trachomatis
,
Bartonella
species,
Francisella tularensis
, and mycobacteria. However, care should be taken when identifying
Chlamydia
spp. and
Bartonella
spp. using polyclonal antibodies, since species of both genera have common antigens which are responsible for cross-reactions.
Persistence of Human Papillomavirus DNA in Benign and (Pre)malignant Skin Lesions from Renal Transplant Recipients
Authors: Berkhout Ron J. M. , Bouwes Bavinck Jan N. , ter Schegget Jan 
Published: March 2020

ABSTRACT
An extremely diverse group of human papillomavirus (HPV) types consisting of epidermodysplasia verruciformis (EV)-associated HPV types and other cutaneous HPV types (e.g., HPV types 2 and 3) is associated with nonmelanoma cancers and benign lesions of the skin. The frequent presence of multiple HPV types in single skin biopsy specimens of renal transplant recipients prompted us to develop PCR techniques for the detection of distinct (sub)groups of genotypically related cutaneous HPV types, i.e., three subgroups of EV-associated HPV types and two groups (A2 and A4) of other cutaneous HPV types. This approach generally allowed a reliable identification of HPV genotypes by direct sequencing of the PCR products, despite the frequent occurrence of multiple infections. The targeted spectrum of HPV types comprises 66 cutaneous HPV types including 21 putative novel HPV types. We also detected 17 putative novel HPV subtypes. We demonstrated that the skin of nearly all renal transplant recipients who developed various benign and (pre)malignant skin lesions was persistently infected with one or more EV-associated HPV types and/or HPV types belonging to groups A2 and A4. The frequency and distribution of EV-associated HPV and HPV types belonging to groups A2 and A4 were similar in biopsy specimens from hyperkeratotic papillomas (77.5%), squamous cell carcinomas (77.8%), and actinic keratoses (67.9%) but appeared to be lower in specimens of basal cell carcinomas (35.7%), benign lesions (38.5%), and clinically normal skin (32.3%). These findings suggest that renal transplant recipients are prone to persistent cutaneous HPV infection. Our data do not support the existence of high-risk cutaneous HPV types.
A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis
Authors: Robertson J. , Guy E. , Andrews N. , Wilske B. , Anda P. , Granström M. , Hauser U. , Moosmann Y. , Sambri V. , Schellekens J. , Stanek G. , Gray J. 
Published: March 2020

ABSTRACT

A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis showed considerable variation in results obtained from tests with a panel of 227 serum samples. Six laboratories used different immunoblot methods, and a wide range of bands was detected in all the assays. Multivariable logistic regression analysis of data from individual laboratories was used to determine the most discriminatory bands for reliable detection of antibodies to
Borrelia burgdorferi
sensu lato. These bands were used to construct individual interpretation rules for the immunoblots used in the six laboratories. Further analysis identified a subset of eight bands, which were important in all the laboratories, although with variations in significance. Possible European rules, all closely related, were formulated from these bands, although there was no single rule that gave high levels of sensitivity and specificity for all the laboratories. This is a reflection of the wide range of methodologies used, especially the use of different species and strains of
B. burgdorferi
sensu lato. The panel of European rules provides a framework for immunoblot interpretation which may be adapted in relation to the characteristics of Lyme borreliosis in local areas.
Accuracy and Precision of Quantitative Calibrated Loops in Transfer of Bronchoalveolar Lavage Fluid
Authors: Jacobs J. A. , De Brauwer E. I. G. B. , Cornelissen E. I. M. , Drent M. 
Published: March 2020

ABSTRACT
Quantitative cultures of bronchoalveolar lavage (BAL) fluid are important in the diagnosis of ventilator-associated pneumonia, and calibrated loops are commonly used to set up these cultures. In this study, the performances of calibrated 0.010- and 0.001-ml loops in the transfer of BAL fluid were determined. Five loops of one lot from seven manufacturers were tested. Calibrations were performed by the gravimetric method (0.010-ml loops) and the colorimetric method (0.001-ml loops). Most of the 0.010-ml loops displayed a precision that was less than 10%, but six of them showed very poor accuracies as they transferred a deficiency (nichrome loops) or an excess (disposable loops) of BAL fluid that exceeded ±10%. The mean maximum and minimum BAL fluid volumes delivered by the 0.010-ml loops differed by a factor 3. The 0.001-ml loops displayed acceptable precision. Five of them showed inaccuracies of ≤±10%, and mean maximum and minimum BAL fluid volumes had a range of a factor of 2. For all loops, the volumes of BAL fluid sampled were larger than the volumes of reagent-grade water sampled. Results of the colony counting experiments confirmed these findings and revealed a high intra-assay variability for the 0.001-ml loops. We conclude that, when BAL fluid samples are cultured with calibrated loops, (i) proper verification of the calibration of these loops is mandatory, (ii) calibrations should be performed with BAL fluid as the test solution, and (iii) borderline quantitative culture results should be interpreted with knowledge of the inaccuracy values of these loops.
Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients
Authors: Caliendo Angela M. , St. George Kirsten , Kao Shaw-Yi , Allega Jessica , Tan Ban-Hock , LaFontaine Robert , Bui Larry , Rinaldo Charles R. 
Published: March 2020

ABSTRACT
The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.
Macrorestriction Fingerprinting of “
Streptococcus milleri
” Group Bacteria by Pulsed-Field Gel Electrophoresis
Authors: Bartie K. L. , Wilson M. J. , Williams D. W. , Lewis M. A. O. 
Published: March 2020

ABSTRACT

Although isolates of the “
Streptococcus milleri
” group (SMG) of bacteria are regarded as members of the commensal microflora of the body, they are frequently encountered in purulent infections from a range of body sites. The genetic diversity of 91 epidemiologically unrelated SMG isolates (including 37 commensal strains and 49 disease-associated strains) was analyzed by macrorestriction fingerprinting (MF). The genomes were digested with
Sma
I and
Apa
I independently, and fragments were resolved by pulsed-field gel electrophoresis. Similarities between banding profiles were determined, and strains were clustered on this basis into dendrograms. In common with other commensal species that have been examined by MF, considerable genetic diversity was revealed. In addition, the clustering of strains tended to support the current taxonomic position of this heterogeneous group. The present study has shown that MF is a powerful tool for characterization of SMG strains and that its use is likely to be of great value in epidemiological and population genetic studies of this group of bacteria.
Comparison of Pulsed-Field Gel Electrophoresis and Coagulase Gene Restriction Profile Analysis Techniques in the Molecular Typing of
Staphylococcus aureus
Authors: Chiou Chien-Shun , Wei Hsiao-Lun , Yang Li-Chu 
Published: March 2020

ABSTRACT

Pulsed-field gel electrophoresis (PFGE) and coagulase gene restriction profile (CRP) analysis techniques were used to analyze 71
Staphylococcus aureus
isolates recovered from nine food-borne disease outbreaks. Twenty-two PFGE profiles and 11 CRPs were identified, with discrimination indices of 0.86 and 0.72, respectively. In addition, the variable regions of the coagulase genes of 39 isolates were sequenced and showed extensive identity, indicating that this is not an efficient alternative for the molecular typing of
S. aureus
.