bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Amino Acid Residues of Anthrax Protective Antigen Involved in Binding with Lethal Factor
Authors: Chauhan Vibha , Bhatnagar Rakesh 
Published: July 2002

ABSTRACT
Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for the translocation of LF to the cytosol. The binding of LF to cell surface receptor-bound PA is a prerequisite for the formation of lethal toxin. It has been hypothesized that hydrophobic residues P184, L187, F202, L203, P205, I207, I210, W226, and F236 of domain 1b of PA play an important role in the binding of PA to LF. These residues are normally buried in the 83-kDA version of PA, PA83, as determined by the crystal structure of PA. However, they become exposed due to the conformational change brought about by the cleavage of PA83 to PA63 by a cell surface protease. Mutation of the above-mentioned residues to alanine resulted in mutant proteins that were able to bind to the cell surface receptors and also to be specifically cleaved by the cellular proteases. All the mutant proteins except the F202A, L203A, P205A, and I207A mutants were able to bind to LF and were also toxic to macrophage cells in combination with LF. It was concluded that residues 202, 203, 205, and 207 of PA are essential for the binding of LF to PA.
Patterns of Epithelial Cell Invasion by Different Species of the
Burkholderia cepacia
Complex in Well-Differentiated Human Airway Epithelia
Authors: Schwab Ute , Leigh Margaret , Ribeiro Carla , Yankaskas James , Burns Kimberly , Gilligan Peter , Sokol Pamela , Boucher Richard 
Published: July 2002

ABSTRACT

Burkholderia cepacia
has emerged as a serious respiratory pathogen in cystic fibrosis (CF) patients. The clinical course of
B. cepacia
infections is variable, but ∼20% of patients eventually succumb to the cepacia syndrome, which is characterized as a fatal necrotizing pneumonia with bacteremia. The mechanisms that permit
B. cepacia
to cause bacteremia are not yet known but probably involve sequential penetration of airway barriers. This study evaluated the abilities of different species of the
B. cepacia
complex, including a strain from the ET12 lineage (BC-7, genomovar III,
cblA
+
), which is associated with most cepacia syndrome fatalities among CF populations, a genomovar IV strain (HI2258), and a genomovar II strain (J-1) to penetrate polarized, well-differentiated human airway epithelial cell cultures. As revealed by light and electron microscopy, all three
B. cepacia
strains tested circumvented the mechanical barriers of mucus and ciliary transport to penetrate the airway epithelium but they used different routes. The BC-7 strain (genomovar III) formed biofilms in close proximity to the apical cell surface, followed by invasion and destruction of epithelial cells. This process involved disruption of the glycocalyx and rearrangements of the actin cytoskeleton. The HI2258 strain (genomovar IV) did not form biofilms, and the majority of bacteria that penetrated the epithelium were located between epithelial cells, suggesting paracytosis. Strain J-1 penetrated the epithelium both by cell destruction and paracytosis. These studies suggest that the distinct invasion pathways employed by
B. cepacia
may account for differences in virulence between
B. cepacia
genomovars.
CD40 Ligand (CD154) Does Not Contribute to Lymphocyte-Mediated Inhibition of VirulentMycobacterium tuberculosiswithin Human Monocytes
Authors: Larkin Rhonda , Benjamin Christopher D. , Hsu Yen-Ming , Li Qing , Zukowski Lynn , Silver Richard F. 
Published: July 2002

ABSTRACTHuman monocytes displayed increased expression of CD40 following infection with virulentMycobacterium tuberculosis. Nevertheless, soluble CD40 ligand (CD40L; also designated CD154) had no effect on the intracellular growth of the organism. Restriction of the intracellular growth ofM. tuberculosisby peripheral blood lymphocytes and antigen-specific CD4+T-cell lines likewise was not reduced by blocking anti-CD40L monoclonal antibody 5c8.
Analysis of DNA Regulatory Elements Required for Expression of theLegionella pneumophilaicmanddotVirulence Genes
Authors: Gal-Mor Ohad , Zusman Tal , Segal Gil 
Published: July 2002

ABSTRACTTo investigate the regulation of theLegionella pneumophila icmanddotgenes required for intracellular growth, a series of nineicm::lacZfusions were constructed. Theseicm::lacZfusions were found to have different levels of expression inL. pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase. When the expression of these fusions inEscherichia coliwas tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those inL. pneumophila. Site-directed and PCR random mutagenesis with theseicm::lacZfusions was used to identify DNA regulatory elements oficmgenes. Fouricmgenes (icmT,icmP,icmQ, andicmM) that had low levels of expression inL. pneumophilawere found to contain a 6-bp sequence (TATACT) essential for their expression. This sequence was shown by primer extension to serve as their −10 promoter elements. A similar sequence, which constitutes the −10 promoter elements of theicmV,icmW, andicmRgenes which had high levels of expression inL. pneumophila, was also identified. In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes. Altogether, 12 regulatory elements, 7 of which constitute the −10 promoter elements of theicmgenes, were found. Even though all theicmanddotgenes are part of one system required forL. pneumophilaintracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors.
Mutations in the CCGTTCACA DnaA Box of
Mycobacterium tuberculosis oriC
That Abolish Replication of
oriC
Plasmids Are Tolerated on the Chromosome
Authors: Dziadek Jaroslaw , Rajagopalan Malini , Parish Tanya , Kurepina Natalia , Greendyke Rebecca , Kreiswirth Barry N. , Madiraju Murty V. V. S. 
Published: July 2002

ABSTRACT

The origin of replication (
oriC
) region in some clinical strains of
Mycobacterium tuberculosis
is a hot spot for IS
6110
elements. To understand how clinical strains with insertions in
oriC
can replicate their DNA, we characterized the
oriC
regions of some clinical strains. Using a plasmid-based
oriC
-dependent replication assay, we showed that IS
6110
insertions that disrupted the DnaA box sequence CCGTTCACA abolished
oriC
activity in
M. tuberculosis
. Furthermore, by using a surface plasmon resonance technique we showed that purified
M. tuberculosis
DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of
oriC
plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain
M. tuberculosis
H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that
M. tuberculosis
strains have evolved mechanisms to tolerate mutations in the
oriC
region and that functional requirements for
M. tuberculosis oriC
replication are different for chromosomes and plasmids.
Identification of a New Class of Cytochrome P450 from a
Rhodococcus
sp
Authors: Roberts Gareth A. , Grogan Gideon , Greter Andy , Flitsch Sabine L. , Turner Nicholas J. 
Published: July 2002

ABSTRACT

A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from
Rhodococcus
sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in
Escherichia coli
, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of
E. coli
was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.
An AraC/XylS Family Member at a High Level in a Hierarchy of Regulators for Phenol-Metabolizing Enzymes inComamonas testosteroniR5
Authors: Teramoto Maki , Ohnishi Kouhei , Harayama Shigeaki , Watanabe Kazuya 
Published: July 2002

ABSTRACTComamonas testosteronistrain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption ofphcTin strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption ofphcTin strain R5 increased the expression ofphcR. The phenol-oxygenating activity was abolished by the disruption ofphcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption ofphcShas been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.
Enterococcus faecalis
3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis
Authors: Sutherlin Autumn , Hedl Matija , Sanchez-Neri Barbara , Burgner John W. , Stauffacher Cynthia V. , Rodwell Victor W. 
Published: July 2002

ABSTRACT

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and
Enterococcus faecalis
genomic DNA to isolate the
mvaS
gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway.
mvaS
was expressed in
Escherichia coli
from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni
2+
-agarose to apparent homogeneity and a specific activity of 10 μmol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa;
s

20,
w

, 5.3). Optimal activity occurred in 2.0 mM MgCl
2
at 37
o
C. The ΔH
a
was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK
a
of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 ± 0.2 and that of covalent acetylation was 0.60 ± 0.02. The

K
m

for the hydrolysis of acetyl-CoA was 10 μM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from
E. faecalis
.
The RuvABC Resolvase Is Indispensable for Recombinational Repair in
sbcB15
Mutants of
Escherichia coli
Authors: Zahradka Davor , Zahradka Ksenija , Petranović Mirjana , Đermić Damir , Brčić-Kostić Krunoslav 
Published: July 2002

ABSTRACT

The RuvABC proteins of
Escherichia coli
play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the
ruv
genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a
recBC sbcBC
background. Genetic analysis presented in this work revealed that the Δ
ruvABC
mutation causes an identical DNA repair defect in UV-irradiated
recBC sbcBC
,
sbcBC
, and
sbcB
strains, indicating that the
sbcB
mutation alone is responsible for the extreme UV sensitivity of
recBC sbcBC ruv
derivatives. In experiments with gamma irradiation and in conjugational crosses, however,
sbcBC
Δ
ruvABC
and
sbcB
Δ
ruvABC
mutants displayed higher recombination proficiency than the
recBC sbcBC
Δ
ruvABC
strain. The frequency of conjugational recombination observed with the
sbcB
Δ
ruvABC
strain was quite similar to that of the Δ
ruvABC
single mutant, indicating that the
sbcB
mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.
The Unique
tuf2
Gene from the Kirromycin Producer
Streptomyces ramocissimus
Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu
Authors: Olsthoorn-Tieleman Lian N. , Fischer Sylvia E. J. , Kraal Barend 
Published: July 2002

ABSTRACT

Streptomyces ramocissimus
, the producer of elongation factor Tu (EF-Tu)-targeted antibiotic kirromycin, contains three divergent
tuf
-like genes, with
tuf1
encoding regular kirromycin-sensitive EF-Tu1; the functions of
tuf2
and
tuf3
are unknown. Analysis of the
tuf
gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of
tuf1
and sometimes of
tuf3
but that
tuf2
was found in
S. ramocissimus
only. The
tuf2
-flanking regions were sequenced, and the two
tuf2
-surrounding open reading frames were shown to be oriented in opposite directions. In vivo transcription analysis of the
tuf2
gene displayed an upstream region with bidirectional promoter activity. The transcription start site of
tuf2
was located approximately 290 nucleotides upstream of the coding sequence. Very small amounts of
tuf2
transcripts were detected in both liquid- and surface-grown cultures of
S. ramocissimus
, consistent with the apparent absence of EF-Tu2 in total protein extracts. The
tuf2
transcript level was not influenced by the addition of kirromycin to exponentially growing cultures. To assess the function of
S. ramocissimus
EF-Tu2, the protein was overexpressed in
Streptomyces coelicolor
LT2. This strain is a J1501 derivative containing His
6
-tagged EF-Tu1 as the sole EF-Tu species, which facilitated the separation of EF-Tu2 from the interfering EF-Tu1.
S. ramocissimus
EF-Tu1 and EF-Tu2 were indistinguishable in their ability to stimulate protein synthesis in vitro and exhibited the same kirromycin sensitivity, which excludes the possibility that EF-Tu2 is directly involved in the kirromycin resistance mechanism of
S. ramocissimus
.
The Chloramphenicol-Inducible
catB
Gene in
Agrobacterium tumefaciens
Is Regulated by Translation Attenuation
Authors: Rogers Elizabeth J. , Rahman M. Sayeedur , Hill Russell T. , Lovett Paul S. 
Published: July 2002

ABSTRACT

Agrobacterium tumefaciens
strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the
catB
gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the
catB
gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible
catA
genes resident in gram-positive bacteria and the inducible
cmlA
gene that confers chloramphenicol resistance in
Pseudomonas
spp.
Minimizing the Workup of Blood Culture Contaminants: Implementation and Evaluation of a Laboratory-Based Algorithm
Authors: Richter S. S. , Beekmann S. E. , Croco J. L. , Diekema D. J. , Koontz F. P. , Pfaller M. A. , Doern G. V. 
Published: July 2002

ABSTRACT

An algorithm was implemented in the clinical microbiology laboratory to assess the clinical significance of organisms that are often considered contaminants (coagulase-negative staphylococci, aerobic and anaerobic diphtheroids,
Micrococcus
spp.,
Bacillus
spp., and viridans group streptococci) when isolated from blood cultures. From 25 August 1999 through 30 April 2000, 12,374 blood cultures were submitted to the University of Iowa Clinical Microbiology Laboratory. Potential contaminants were recovered from 495 of 1,040 positive blood cultures. If one or more additional blood cultures were obtained within ±48 h and all were negative, the isolate was considered a contaminant. Antimicrobial susceptibility testing (AST) of these probable contaminants was not performed unless requested. If no additional blood cultures were submitted or there were additional positive blood cultures (within ±48 h), a pathology resident gathered patient clinical information and made a judgment regarding the isolate's significance. To evaluate the accuracy of these algorithm-based assignments, a nurse epidemiologist in approximately 60% of the cases performed a retrospective chart review. Agreement between the findings of the retrospective chart review and the automatic classification of the isolates with additional negative blood cultures as probable contaminants occurred among 85.8% of 225 isolates. In response to physician requests, AST had been performed on 15 of the 32 isolates with additional negative cultures considered significant by retrospective chart review. Agreement of pathology resident assignment with the retrospective chart review occurred among 74.6% of 71 isolates. The laboratory-based algorithm provided an acceptably accurate means for assessing the clinical significance of potential contaminants recovered from blood cultures.
Ralstonia pickettii
Traced in Blood Culture Bottles
Authors: Boutros Névine , Gonullu Nevriye , Casetta Anne , Guibert Michèle , Ingrand Didier , Lebrun Léa 
Published: July 2002

ABSTRACT

Over a 9-month period, 14 strains of
Ralstonia pickettii
were isolated from various biological samples inoculated in a blood culture medium. Molecular epidemiological investigation confirmed the relatedness of the strains. The source of the contamination proved to be the blood culture bottle caps.
Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins
Authors: Buonocore Linda , Blight Keril J. , Rice Charles M. , Rose John K. 
Published: July 2002

ABSTRACT
We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These ΔG viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.
Analysis of a Temperature-Sensitive Mutant Rotavirus Indicates that NSP2 Octamers Are the Functional Form of the Protein
Authors: Taraporewala Zenobia F. , Schuck Peter , Ramig Robert F. , Silvestri Lynn , Patton John T. 
Published: July 2002

ABSTRACT

Evidence that NSP2 plays a role in packaging and replication comes from studies on
tsE
(
1400
), a rotavirus mutant with a temperature-sensitive (
ts
) lesion in the NSP2 gene. Cells infected with
tsE
and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty. Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the
ts
phenotype of
tsE
. To gain insight into the effect of the mutation on the octameric structure and biochemical activities of
tsE
NSP2, the protein was expressed in bacteria and purified to homogeneity. Analytical ultracentrifugation showed that
tsE
NSP2 formed octamers which, like those formed by wild-type (
wt
) NSP2, undergo conformational change into more compact structures upon binding of nucleotides. However, exposure to Mg
2+
and the nonpermissive temperature caused disruption of the
tsE
octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with
wt
octamers. Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of
tsE
NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature. In contrast, these activities for
wt
NSP2 were higher at the nonpermissive temperature. Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication. The propensity of
tsE
NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.