bluuebunny/crossref_metadata_2025_split huggingface dataset:
Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na
+
(Li
+
)/H
+
Antiporter Activity on
Escherichia coli
: Characterization of the Recovered Genes and the Corresponding Gene Products
Authors: Majernı́k Alan , Gottschalk Gerhard , Daniel Rolf 
Published: July 2002

ABSTRACT

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na
+
(Li
+
)/H
+
antiporter activity on the antiporter-deficient
Escherichia coli
strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive
E. coli
clones were obtained during the initial screening of 1,480,000 recombinant
E. coli
strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li
+
-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na
+
/H
+
antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of
E. coli
K-12 containing
nhaA
,
nhaR
, and
gef
. This region is flanked by highly conserved insertion elements. The sequence identity with
E. coli
decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from
E. coli
. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of
E. coli
and enabled the antiporter-deficient
E. coli
mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na
+
/H
+
antiporter activity in everted membrane vesicles that were derived from the
E. coli
strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The

K
m

values of both antiporters for Na
+
were approximately 10-fold higher than the values for Li
+
.
Analysis of Early Promoters of the
Bacillus
Bacteriophage GA-1
Authors: Horcajadas José A. , Meijer Wilfried J. J. , Rojo Fernando , Salas Margarita 
Published: July 2002

ABSTRACT

Bacteriophage GA-1, which infects
Bacillus
sp. strain G1R, is evolutionarily related to phage φ29, which infects
Bacillus subtilis
. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of φ29 or related phages. These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.
Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed
oriT
Cleavage In Vivo
Authors: Karl Wolfgang , Bamberger Martina , Zechner Ellen L. 
Published: July 2002

ABSTRACT

The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (
oriT
) in vivo was investigated. As expected, the cleavage reaction was not detected in
Escherichia coli
cells expressing
tral
and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at
oriT
in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1
oriT
constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations,
traY
was dispensable for mobilization in the presence of
traM
, but mobilization did not occur in the absence of both
traM
and
traY
. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.
The Hook Gene (
flgE
) Is Expressed from the
flgBCDEF
Operon in
Rhodobacter sphaeroides
: Study of an
flgE
Mutant
Authors: Ballado Teresa , Camarena Laura , González-Pedrajo Bertha , Silva-Herzog Eugenia , Dreyfus Georges 
Published: July 2002

ABSTRACT

In this work we identified the
flgE
gene encoding the flagellar hook protein from
Rhodobacter sphaeroides
. Our results show that this gene is part of a flagellar cluster that includes the genes
flgB, flgC, flgD, flgE
, and
flgF
. Two different types of mutants in the
flgE
gene were isolated, and both showed a Fla
−
phenotype, indicating the functionality of this sequence. Complementation studies of these mutant strains suggest that
flgE
is included in a single transcriptional unit that starts in
flgB
and ends in
flgF
. In agreement with this possibility, a specific transcript of approximately 3.5 kb was identified by Northern blot. This mRNA is large enough to represent the complete
flgBCDEF
operon. FlgE showed a relatively high proline content; in particular, a region of 12 amino acids near the N terminus, in which four prolines were identified. Cells expressing a mutant FlgE protein lacking this region showed abnormal swimming behavior, and their hooks were curved. These results suggest that this region is involved in the characteristic quaternary structure of the hook of
R. sphaeroides
and also imply that a straight hook, or perhaps the rigidity associated with this feature, is important for an efficient swimming behavior in this bacterium.
The
secE
Gene of
Helicobacter pylori
Authors: Médigue Claudine , Wong Benjamin Chun-Yu , Lin Marie Chia-Mi , Bocs Stéphanie , Danchin Antoine 
Published: July 2002

ABSTRACT

Despite extensive annotation by two independent teams, the
Helicobacter pylori
genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing
secE
component of the major secretion machinery of
Helicobacter pylori
.
Identification and Characterization of
pvuA
, a Gene Encoding the Ferric Vibrioferrin Receptor Protein in
Vibrio parahaemolyticus
Authors: Funahashi Tatsuya , Moriya Kaoru , Uemura Sachi , Miyoshi Shin-ichi , Shinoda Sumio , Narimatsu Shizuo , Yamamoto Shigeo 
Published: July 2002

ABSTRACT

We previously reported that
Vibrio parahaemolyticus
expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, Y. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of
V. parahaemolyticus
WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene (
psuA
) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene (
pvuA
) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by
pvuA
displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of
Morganella morganii
. Primer extension and Northern blot analyses indicated that
psuA
and
pvuA
constitute an operon which is transcribed from a Fur-repressed promoter upstream of
psuA
. The product of the
pvuA
gene and its function were confirmed by generating a
pvuA
-disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream
psuA
gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin.
TrwD, the Hexameric Traffic ATPase Encoded by Plasmid R388, Induces Membrane Destabilization and Hemifusion of Lipid Vesicles
Authors: Machón Cristina , Rivas Susana , Albert Armando , Goñi Félix M. , de la Cruz Fernando 
Published: July 2002

ABSTRACT

TrwD, a hexameric ATP hydrolase encoded by plasmid R388, is a member of the PulE/VirB11 protein superfamily of traffic ATPases. It is essential for plasmid conjugation, particularly for expression of the conjugative W pilus. In the present study, we analyzed the effects that TrwD produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts. TrwD induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monolayers in the presence of calcium. It also induced extensive leakage of the vesicular aqueous contents. A point mutant of TrwD with a mutation in the P loop of the nucleotide-binding region (K203Q) that lacks both ATPase activity and the ability to support conjugation showed the same behavior as native TrwD in all of these processes, which were independent of the presence of ATP. Structure prediction methods revealed a close similarity to
Helicobacter pylori
protein HP0525, another member of the PulE/VirB11 family, whose crystal structure is known. The interpretation of our data in the light of this structure is that TrwD interacts with the lipid bilayer through hydrophobic regions in its N-terminal domain, which leads to a certain degree of membrane destabilization. TrwD appears to be a part of the conjugation machinery that interacts with the membranous systems in order to facilitate DNA transfer in bacteria.
Membrane Topology of theStreptococcus pneumoniaeFtsW Division Protein
Authors: Gérard Philippe , Vernet Thierry , Zapun André 
Published: July 2002

ABSTRACTThe topology of FtsW fromStreptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm.
Population Genetic Structure of
Legionella pneumophila
Inferred from RNA Polymerase Gene (
rpoB
) and DotA Gene (
dotA
) Sequences
Authors: Ko Kwan Soo , Lee Hae Kyung , Park Mi-Yeoun , Park Man-Suk , Lee Keun-Hwa , Woo So-Yon , Yun Yeo-Jun , Kook Yoon-Hoh 
Published: July 2002

ABSTRACT

The population structure of
Legionella pneumophila
was studied by using partial RNA polymerase gene (
rpoB
) and DotA gene (
dotA
) sequences. Trees inferred from
rpoB
sequences showed that two subspecies of
L. pneumophila
,
Legionella pneumophila
subsp.
pneumophila
and
Legionella pneumophila
subsp.
fraseri
, were clearly separated genetically. In both
rpoB
and
dotA
trees, 79 Korean isolates used in this study constituted six clonal populations, four of which (designated subgroups P-I to P-IV) were identified in
L. pneumophila
subsp.
pneumophila
and two of which (designated subgroups F-I and F-II) were identified in
L. pneumophila
subsp.
fraseri
. Although the relationships among subgroups were not identical, such subgrouping was congruent between the
rpoB
and
dotA
trees. Type strains of several serogroups did not belong to any subgroup, presumably because isolates similar to these strains were not present among our local sample of the population. There was evidence that horizontal gene transfer or recombination had occurred within
L. pneumophila
. Contrary to the phylogeny from
rpoB
and the taxonomic context, subgroups P-III and P-IV of
L. pneumophila
subsp.
pneumophila
proved to be closely related to those of
L. pneumophila
subsp.
fraseri
or showed a distinct clustering in the
dotA
tree. It can be inferred that
dotA
of subgroups P-III and P-IV has been transferred horizontally from other subspecies. The diverse distribution of serogroup 1 strains through the gene trees suggests that surface antigen-coding genes that determine serogroup can be exchanged. Thus, it can be inferred that genetic recombination has been important in the evolution of
L. pneumophila
.
Rapid Identification of Laboratory Contamination with
Mycobacterium tuberculosis
Using Variable Number Tandem Repeat Analysis
Authors: Gascoyne-Binzi Deborah M. , Barlow Rachael E. L. , Frothingham Richard , Robinson Grant , Collyns Timothy A. , Gelletlie Ruth , Hawkey Peter M. 
Published: July 2002

ABSTRACT

Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination. Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize. Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible. The digital profile is easily manipulated in a database. We undertook a retrospective study of
Mycobacterium tuberculosis
isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system. VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data. We found 36 distinct VNTR profiles in cultures from 144 patients. Three common VNTR profiles accounted for 45% of true-positive cases. By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected. These nine incidents resulted in 34 false-positive cultures for 29 patients. False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment. Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff. Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events. VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures. This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures. In addition, areas where existing procedures may be improved can be identified.
Magnetic Immuno-PCR Assay with Inhibitor Removal for Direct Detection of
Helicobacter pylori
in Human Feces
Authors: Monteiro Lurdes , Gras Nathalie , Megraud Francis 
Published: July 2002

ABSTRACT

A PCR protocol was developed to detect
Helicobacter pylori
in human stool specimens. This protocol was based on the association of a magnetic immuno-PCR assay with a technique to remove inhibitors (agarose-embedded DNA preparation). Of the 47
H. pylori
-positive and 57
H. pylori
-negative patients included in this study, 38 were positive and 66 were negative by this new protocol. The sensitivity, specificity, and predictive values for a positive or a negative result were 80.9% (95% confidence interval [CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI, 88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.
Genetic Diversity among
Mycobacterium bovis
Isolates: a Preliminary Study of Strains from Animal and Human Sources
Authors: Sales M. P. U. , Taylor G. M. , Hughes S. , Yates M. , Hewinson G. , Young D. B. , Shaw R. J. 
Published: July 2002

ABSTRACT

Mycobacterium bovis
has the broadest host range of species in the
Mycobacterium tuberculosis
complex and is responsible for disease in humans and diverse animal species. We report on genotypic differences at multiple loci among 13 isolates derived from a range of human and animal infections. All isolates were classified as
M. bovis
by phenotypic analysis but could be subdivided into five distinct genotypes based on polymorphisms at the
pncA
and
oxyR
loci, the status of the RD5 deletion region, and the spoligotype pattern. These findings suggest the existence of a spectrum of strains with genotypic characteristics between those of
M. tuberculosis
and
M. bovis
.
Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity
Authors: Gomez Arley , Mve-Obiang Armand , Vray Bernard , Rudnicka Wieslawa , Shamputa Isdore C. , Portaels Françoise , Meyers Wayne M. , Fonteyne Pierre-Alain , Realini Laurence 
Published: July 2002

ABSTRACTPhospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused byClostridium perfringensandListeria monocytogenes. Previous studies have reported multiple copies ofplcgenes homologous toPseudomonas aeruginosa plcHandplcNgenes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes ofMycobacterium tuberculosis, M. marinum, M. bovis, andM. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences ofM. kansasiiandM. marinumhomologous to the genes encoding phospholipase C fromM. tuberculosisandM. ulceranswere identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected inM. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis,andM. kansasiimycobacteria with phospholipase C activity, but not inM. fortuitum. No hemolytic activity was detected inM. smegmatis, M. gordonae, andM. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.
Specific Detection of
Pasteurella multocida
in Chickens with Fowl Cholera and in Pig Lung Tissues Using Fluorescent rRNA In Situ Hybridization
Authors: Mbuthia Paul Gichohi , Christensen Henrik , Boye Mette , Petersen Kamille Majken Dumong , Bisgaard Magne , Nyaga Phillip Njeru , Olsen John Elmerdahl 
Published: July 2002

ABSTRACT

A
Pasteurella multocida
species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated
P. multocida
from other bacterial species of the families
Pasteurellaceae
and
Enterobacteriaceae
and also from divergent species of the order
Cytophagales
(except biovar 2 strains of
Pasteurella avium
and
Pasteurella canis,
which have high 16S rRNA similarity to
P. multocida
). The potential of the probe for specific identification and differentiation of
P. multocida
was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues
P. multocida
cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected
P. multocida
was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of
P. multocida
in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to
P. multocida
.
Reproducibility of
Bordetella pertussis
Genomic DNA Fragments Generated by
Xba
I Restriction and Resolved by Pulsed-Field Gel Electrophoresis
Authors: Hardwick Terri H. , Plikaytis Brian , Cassiday Pamela K. , Cage Gary , Peppler Mark S. , Shea Deborah , Boxrud David , Sanden Gary N. 
Published: July 2002

ABSTRACT

The intra- and interlaboratory variabilities of the molecular size measurements of each DNA fragment contributing to three pulsed-field gel electrophoresis (PFGE) profiles were assessed, as were the reproducibilities of the entire PFGE profiles for three
Bordetella pertussis
strains. The major source of variability within a laboratory occurred between subcultures rather than within gels or between gels. Each PFGE profile was generated reproducibly and was objectively defined by the molecular sizes of its composite fragments. A strain or profile most suitable for use as an internal reference standard was identified.
Ca3 Fingerprinting of
Candida albicans
Isolates from Human Immunodeficiency Virus-Positive and Healthy Individuals Reveals a New Clade in South Africa
Authors: Blignaut Elaine , Pujol Claude , Lockhart Shawn , Joly Sophie , Soll David R. 
Published: July 2002

ABSTRACT

To examine the question of strain specificity in oropharyngeal candidiasis associated with human immunodeficiency virus (HIV) infection, oral samples were collected from 1,196 HIV-positive black South Africans visiting three clinics and 249
Candida albicans
isolates were selected for DNA fingerprinting with the complex DNA fingerprinting probe Ca3. A total of 66
C. albicans
isolates from healthy black South Africans and 46 from healthy white South Africans were also DNA fingerprinted as controls. Using DENDRON software, a cluster analysis was performed and the identified groups were compared to a test set of isolates from the United States in which three genetic groups (I, II, and III) were previously identified by a variety of genetic fingerprinting methods. All of the characterized South African collections (three from HIV-positive black persons, two from healthy black persons, and one from healthy white persons) included group I, II, and III isolates. In addition, all South African collections included a fourth group (group SA) completely absent in the U.S. collection. The proportion of group SA isolates in HIV-positive and healthy black South Africans was 53% in both cases. The proportion in healthy white South Africans was 33%. In a comparison of HIV-positive patients with and without oropharyngeal symptoms of infection, the same proportions of group I, II, III, and SA isolates were obtained, indicating no shift to a particular group on infection. However, by virtue of its predominance as a commensal and in infections, group SA must be considered the most successful in South Africa. Why group SA isolates represent 53 and 33% of colonizing strains in black and white South Africans and are absent in the U.S. collection represents an interesting epidemiological question.