bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Stx2-Specific Monoclonal Antibodies Prevent Systemic Complications of
Escherichia coli
O157:H7 Infection
Authors: Mukherjee Jean , Chios Kerry , Fishwild Dianne , Hudson Deborah , O'Donnell Susan , Rich Stephen M. , Donohue-Rolfe Arthur , Tzipori Saul
Published: July 2002

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic
Escherichia coli
(EHEC), such as
E. coli
O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing
E. coli
O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and κ light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1κ A-subunit-specific and 3 IgG1κ and 1 IgG3κ B-subunit-specific antibodies. Six IgG1κ A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1κ B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of >95% activity of 1 ng of Stx2 in the presence of 0.04 μg of HuMAb in vitro and significant prolongation of survival of mice given 50 μg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with
E. coli
O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including
E. coli
O157:H7.
In Vitro and In Vivo Characterization of aBordetella bronchisepticaMutant Strain with a Deep Rough Lipopolysaccharide Structure
Authors: Sisti Federico , Fernández Julieta , Rodríguez María Eugenia , Lagares Antonio , Guiso Nicole , Hozbor Daniela Flavia
Published: July 2002

Bordetella bronchisepticais closely related toBordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research onBordetellahas been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) duringB. bronchisepticainfection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutantB. bronchisepticaLP39 on thewaaCgene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although inB. bronchisepticaLP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS ofB. bronchisepticaLP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, althoughB. bronchisepticaLP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form ofB. bronchisepticaLPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from humanB. bronchisepticachronic infections, the possibility that this phenotype arises as a consequence of selection pressure within the host at a late stage of the infection process is discussed.
Expression of thePlasmodium falciparumImmunodominant Epitope (NANP)4on the Surface ofSalmonella entericaUsing the Autotransporter MisL
Authors: Ruiz-Pérez Fernando , León-Kempis Rocío , Santiago-Machuca Araceli , Ortega-Pierres Guadalupe , Barry Eileen , Levine Myron , González-Bonilla César
Published: July 2002

Gram-negative bacterial proteins which are exported from the cytosol to the external environment by the type V secretion system are also known as autotransporters. Once translocated to the periplasmic compartment by thesec-dependent general secretory pathway, their C-terminal domain forms a pore through which the N-terminal domain travels to the outer membrane without the need of other accessory proteins. MisL (protein of membrane insertion and secretion) is a protein of unknown function located in the pathogenicity island SPI-3 ofSalmonella entericaand classified as an autotransporter due to its high homology toEscherichia coliAIDA-I. In the present work, the MisL C-terminal translocator domain was used to display the immunodominant B-cell epitope of the circumsporozoite protein (CSP) fromPlasmodium falciparumon the surface ofSalmonella entericaserovar Typhimurium (serovar Typhimurium SL3261) and serovar Typhi (serovar Typhi CVD 908). The MisL β domain was predicted by alignment with AIDA-I, amplified from serovar Typhimurium SL3261, cloned in a plasmid fused to four repeats of the tetrapeptide NANP behind theEscherichia coliheat-labile enterotoxin B subunit signal peptide to ensure periplasmic traffic, and expressed under the control of the anaerobically induciblenirBpromoter. The fusion protein was translocated to the outer membrane of both bacterial strains, although the foreign epitope was displayed more efficiently in serovar Typhimurium SL3261, which elicited a better specific antibody response in BALB/c mice. More importantly, antibodies were able to recognize the native CSP inP. falciparumsporozoites. These results confirm that MisL is indeed an autotransporter and that it can be used to express foreign immunogenic epitopes on the surface of gram-negative bacteria.
Identification of Genes Encoding Exported
Mycobacterium tuberculosis
Proteins Using a Tn
552′phoA
In Vitro Transposition System
Authors: Braunstein Miriam , Griffin Thomas J. , Kriakov Jordan I. , Friedman Sarah T. , Grindley Nigel D. F. , Jacobs William R.
Published: July 2002

Secreted and cell envelope-associated proteins are important to both
Mycobacterium tuberculosis
pathogenesis and the generation of protective immunity to
M. tuberculosis
. We used an in vitro Tn
552′phoA
transposition system to identify exported proteins of
M. tuberculosis
. The system is simple and efficient, and the transposon inserts randomly into target DNA.
M. tuberculosis
genomic libraries were targeted with Tn
552′phoA
transposons, and these libraries were screened in
M. smegmatis
for active PhoA translational fusions. Thirty-two different
M. tuberculosis
open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the
M. tuberculosis
databases. This collection of exported proteins includes factors that are known to participate in the immune response of
M. tuberculosis
and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.
2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by
Rhodopseudomonas palustris
Authors: Pelletier Dale A. , Harwood Caroline S.
Published: July 2002

A gene,
badH
, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium
Rhodopseudomonas palustris
. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by
R. palustris
. The 2-hydroxychc-CoA dehydrogenase activity encoded by
badH
was dependent on the presence of NAD
+
; no activity was detected with NADP
+
as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent

K
m

values of 10 and 200 μM for 2-hydroxychc-CoA and NAD
+
, respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown
R. palustris
cell extracts. The active form of the enzyme is a homotetramer.
badH
was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar
R. palustris badH
mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by
R. palustris
. This compound had previously been shown not to be formed by
Thauera aromatica
, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the
R. palustris
pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by
T. aromatica
and thus may serve as a useful indicator of an
R. palustris
-type benzoate degradation pathway.
A Membrane-Bound Flavocytochrome
c
-Sulfide Dehydrogenase from the Purple Phototrophic Sulfur Bacterium
Ectothiorhodospira vacuolata
Authors: Kostanjevecki Vesna , Brigé Ann , Meyer Terrance E. , Cusanovich Michael A. , Guisez Yves , Van Beeumen Jozef
Published: July 2002

The amino acid sequence of
Ectothiorhodospira vacuolata
cytochrome
c
-552, isolated from membranes with
n
-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of
Chlorobium limicola
f. sp.
thiosulfatophilum
flavocytochrome
c
-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against
Chromatium vinosum
FCSD. When aligned according to the three-dimensional structure of
Chromatium
FCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (
C. vinosum
numbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of
E. vacuolata
FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.
Identification, Cloning, and Initial Characterization of
rot
, a Locus Encoding a Regulator of Virulence Factor Expression in
Staphylococcus aureus
Authors: McNamara Peter J. , Milligan-Monroe Kathy C. , Khalili Shirin , Proctor Richard A.
Published: July 2002

A chromosomal insertion of transposon Tn
917
partially restores the expression of protease and alpha-toxin activities to PM466, a genetically defined
agr
-null derivative of the wild-type
Staphylococcus aureus
strain RN6390. In co-transduction experiments, transposon-encoded erythromycin resistance and a protease- and alpha-toxin-positive phenotype are transferred at high frequency from mutant strains to
agr
-null strains of
S. aureus
. Southern analysis of chromosomal DNA and sequence analysis of DNA flanking the Tn
917
insertion site in mutant strains revealed that the transposon interrupted a 498-bp open reading frame (ORF). Similarity searches using a conceptual translation of the ORF identified a region of homology to the known staphylococcal global regulators AgrA and SarA. To verify that the mutant allele conferred the observed phenotype, a wild-type allele of the mutant gene was introduced into the genome of a mutant strain by homologous recombination. The resulting isolates had a restored
agr
-null phenotype. Virulence factor gene expression in mutant, restored mutant, and wild-type strains was quantified by measuring alpha-toxin activity in culture supernatant fluids and by Northern analysis of the alpha-toxin transcript. We named this ORF
rot
(for repressor of toxins) (GenBank accession no.
AF189239
) because of the activity associated with
rot
::Tn
917
mutant strains.
A Redox-Responsive Regulator of Photosynthesis Gene Expression in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Li Hong , Sherman Louis A.
Published: July 2002

We have identified genes in the unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 that are involved with redox control of photosynthesis and pigment-related genes. The genes,
rppA
(sll0797) and
rppB
(sll0798), represent a two-component regulatory system that controls the synthesis of photosystem II (PSII) and PSI genes, in addition to photopigment-related genes.
rppA
(regulator of photosynthesis- and photopigment-related gene expression) and
rppB
exhibit strong sequence similarity to prokaryotic response regulators and histidine kinases, respectively. In the wild type, the steady-state mRNA levels of PSII reaction center genes increased when the plastoquinone (PQ) pool was oxidized and decreased when the PQ pool was reduced, whereas transcription of the PSI reaction center genes was affected in an opposite fashion. Such results suggested that the redox poise of the PQ pool is critical for regulation of the photosystem reaction center genes. In Δ
rppA
, an insertion mutation of
rppA
, the PSII gene transcripts were highly up-regulated relative to the wild type under all redox conditions, whereas transcription of phycobilisome-related genes and PSI genes was decreased. The higher transcription of the
psbA
gene in Δ
rppA
was manifest by higher translation of the D1 protein and a concomitant increase in O
2
evolution. The results demonstrated that RppA is a regulator of photosynthesis- and photopigment-related gene expression, is involved in the establishment of the appropriate stoichiometry between the photosystems, and can sense changes in the PQ redox poise.
TrfA-Dependent Inner Membrane-Associated Plasmid RK2 DNA Synthesis and Association of TrfA with Membranes of Different Gram-Negative Hosts
Authors: Banack Trevor , Kim Peter D. , Firshein William
Published: July 2002

TrfA, the replication initiator protein of broad-host-range plasmid RK2, was tested for its ability to bind to the membrane of four different gram-negative hosts in addition to
Escherichia coli
:
Pseudomonas aeruginosa
,
Pseudomonas putida
,
Salmonella enterica
serovar Typhimurium, and
Rhodobacter sphaeroides
. Cells harboring TrfA-encoding plasmids were fractionated into soluble, inner membrane, and outer membrane fractions. The fractions were subjected to Western blotting, and the blots were probed with antibody to the TrfA proteins. TrfA was found to fractionate with the cell membranes of all species tested. When the two membrane fractions of these species were tested for their ability to synthesize plasmid DNA endogenously (i.e., without added template or enzymes), only the inner membrane fraction was capable of extensive synthesis that was inhibited by anti-TrfA antibody in a manner similar to that of the original host species,
E. coli
. In addition, although DNA synthesis did occur in the outer membrane fraction, it was much less extensive than that exhibited by the inner membrane fraction and only slightly affected by anti-TrfA antibody. Plasmid DNA synthesized by the inner membrane fraction of one representative species,
P. aeruginosa
, was characteristic of supercoil and intermediate forms of the plasmid. Extensive DNA synthesis was observed in the soluble fraction of another representative species,
R. sphaeroides
, but it was completely unaffected by anti-TrfA antibody, suggesting that such synthesis was due to repair and/or nonspecific chain extension of plasmid DNA fragments.
Bacillus subtilis
YvrK Is an Acid-Induced Oxalate Decarboxylase
Authors: Tanner Adam , Bornemann Stephen
Published: July 2002

Bacillus subtilis
has been shown to express a cytosolic oxalate decarboxylase (EC
4.1.1.2
). The enzyme was induced in acidic growth media, particularly at pH 5.0, but not by oxalate. The enzyme was purified, and N-terminal sequencing identified the protein to be encoded by
yvrK
. The role of the first oxalate decarboxylase to be identified in a prokaryote is discussed.
Identification of Two Novel
hrp
-Associated Genes in the
hrp
Gene Cluster of
Xanthomonas oryzae
pv. oryzae
Authors: Zhu Weiguang , MaGbanua Mark M. , White Frank F.
Published: July 2002

We have cloned a
hrp
gene cluster from
Xanthomonas oryzae
pv. oryzae. Bacteria with mutations in the
hrp
region have reduced growth in rice leaves and lose the ability to elicit a hypersensitive response (HR) on the appropriate resistant cultivars of rice and the nonhost plant tomato. A 12,165-bp portion of nucleotide sequence from the presumed left end and extending through the
hrpB
operon was determined. The region was most similar to
hrp
genes from
Xanthomonas campestris
pv. vesicatoria and
Ralstonia solanacearum
. Two new
hrp
-associated loci, named
hpa1
and
hpa2
, were located beyond the
hrpA
operon. The
hpa1
gene encoded a 13-kDa glycine-rich protein with a composition similar to those of harpins and PopA. The product of
hpa2
was similar to lysozyme-like proteins. Perfect PIP boxes were present in the
hrpB
and
hpa1
operons, while a variant PIP box was located upstream of
hpa2
. A strain with a deletion encompassing
hpa1
and
hpa2
had reduced pathogenicity and elicited a weak HR on nonhost and resistant host plants. Experiments using single mutations in
hpa1
and
hpa2
indicated that the loss of
hpa1
was the principal cause of the reduced pathogenicity of the deletion strain. A 1,519-bp insertion element was located immediately downstream of
hpa2
. Hybridization with
hpa2
indicated that the gene was present in all of the strains of
Xanthomonas
examined. Hybridization experiments with
hpa1
and IS
1114
indicated that these sequences were detectable in all strains of
X. oryzae
pv. oryzae and some other
Xanthomonas
species.
Molecular Characterization of Two-Component Systems of
Helicobacter pylori
Authors: Beier Dagmar , Frank Rainer
Published: July 2002

Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogen
Helicobacter pylori
deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3′ end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.
Site-Specific Serine Incorporation by Lif and Epr into Positions 3 and 5 of the Staphylococcal Peptidoglycan Interpeptide Bridge
Authors: Ehlert Kerstin , Tschierske Martin , Mori Claudio , Schröder Werner , Berger-Bächi Brigitte
Published: July 2002

The FemAB-like factors Lif and Epr confer resistance to glycylglycine endopeptidases lysostaphin and Ale-1, respectively, by incorporating serine residues into the staphylococcal peptidoglycan interpeptide bridges specifically at positions 3 and 5. This required the presence of FemA and/or FemB, in contrast to earlier postulations.
l
-Threonine Export: Use of Peptides To Identify a New Translocator from
Corynebacterium glutamicum
Authors: Simic Petra , Sahm Hermann , Eggeling Lothar
Published: July 2002

Bacterial mechanisms for the uptake of peptides and their hydrolysis to amino acids are known in great detail, whereas much less is known about the fates of the peptide-derived amino acids. We show that the addition of
l
-threonine-containing di- or tripeptides results in reduction of the growth of
Corynebacterium glutamicum
, with concomitant high intracellular accumulation of
l
-threonine to up to 130 mM. Using transposon mutagenesis and isolation of mutants with increased Thr peptide sensitivity, nine open reading frames (ORFs) were identified, almost all encoding hypothetical proteins of unknown function. Three ORFs encode membrane proteins. Their individual functional characterizations in the wild-type background led to the identification of
thrE
. Upon
thrE
overexpression, growth is no longer sensitive to the presence of the Thr peptide, and
l
-threonine is exported at a rate of 3.8 nmol min
−1
mg of dry weight
−1
, whereas the rate of export of a
thrE
inactivation mutant is reduced to 1.1 nmol min
−1
mg of dry weight
−1
. In addition to
l
-threonine,
l-
serine is also a substrate for the exporter. The exporter exhibits nine predicted transmembrane-spanning helices with long charged C and N termini and with an amphipathic helix present within the N terminus. All these data suggest that the carrier encoded by
thrE
serves to export small molecules such as
l
-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in
Mycobacterium tuberculosis
and
Streptomyces coelicolor
.
Characterization of Mutations in the
metY
-
nusA
-
infB
Operon That Suppress the Slow Growth of a Δ
rimM
Mutant
Authors: Bylund Göran O. , Lövgren J. Mattias , Wikström P. Mikael
Published: July 2002

The RimM protein in
Escherichia coli
is associated with free 30S ribosomal subunits but not with 70S ribosomes. A Δ
rimM
mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits. The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA. Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a Δ
rimM
mutant by increasing the expression of
rbfA
. Nine of these mutations were in the
nusA
gene, which is located upstream from
rbfA
in the
metY-nusA-infB
operon; three mutations deleted the transcriptional terminator between
infB
and
rbfA
; one was an insertion of IS
2
in
infB
, creating a new promoter for
rbfA
; and one was a duplication, placing a second copy of
rbfA
downstream from a promoter for the
yhbM
gene. Two of the
nusA
mutations were identical, while another mutation (
nusA98
) was identical to a previously isolated mutation,
nusA11
, shown to decrease termination of transcription. The different
nusA
mutations were found to increase the expression of
rbfA
by increasing the read-through of two internal transcriptional terminators located just downstream from the
metY
gene and that of the internal terminator preceding
rbfA
. Induced expression of the
nusA
+
gene from a plasmid in a
nusA
+
strain decreased the read-through of the two terminators just downstream from
metY
, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the
metY-nusA-infB
operon expression is these terminators. All of the
nusA
mutations produced temperature-sensitive phenotypes of
rimM
+
strains. The
nusA
gene has previously been shown to be essential at 42°C and below 32°C. Here, we show that
nusA
is also essential at 37°C.
Novel Form of ClpB/HSP100 Protein in the Cyanobacterium
Synechococcus
Authors: Eriksson Mats-Jerry , Schelin Jenny , Miskiewicz Ewa , Clarke Adrian K.
Published: July 2002

Synechococcus
sp. strain PCC 7942 has a second
clpB
gene that encodes a 97-kDa protein with novel features. ClpBII is the first ClpB not induced by heat shock or other stresses; it is instead an essential, constitutive protein. ClpBII is unable to complement ClpBI function for acquired thermotolerance. No truncated ClpBII version is normally produced, unlike other bacterial forms, while ectopic synthesis of a putative truncated ClpBII dramatically decreased cell viability.
Genomic Sequence and Transcriptional Analysis of a 23-Kilobase Mycobacterial Linear Plasmid: Evidence for Horizontal Transfer and Identification of Plasmid Maintenance Systems
Authors: Le Dantec Corinne , Winter Nathalie , Gicquel Brigitte , Vincent Véronique , Picardeau Mathieu
Published: July 2002

Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from
Mycobacterium celatum
, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in both
M. celatum
pCLP and the
Mycobacterium tuberculosis
chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (
par
operon) and postsegregational (
pem
operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP.
The
Ralstonia eutropha
PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production
Authors: York Gregory M. , Stubbe JoAnne , Sinskey Anthony J.
Published: July 2002

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the
Ralstonia eutropha
PhaR protein on the regulation of PhaP accumulation.
R. eutropha
strains with
phaR
,
phaC
, and/or
phaP
deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the
phaC
deletion strain accumulated no PhaP, as expected. In contrast, both the
phaR
and the
phaR phaC
deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the
R. eutropha phaR
,
phaP
, and PHA biosynthesis (
phaCAB
) genes into a heterologous system,
Escherichia coli
, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of
phaR
caused a decrease in PHA yields, and a
phaR phaP
deletion strain exhibited a more severe PHA defect than a
phaP
deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.
An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions inEscherichia coli
Authors: Brantl Sabine , Wagner E. Gerhart H.
Published: July 2002

Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and theinc18family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported byEscherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivolacZreporter gene assays showed that the antisense RNAs of these plasmids can inhibitlacZexpression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in anE. coliin vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g.,Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.
A Membrane-Bound Archaeal Lon Protease Displays ATP-Independent Proteolytic Activity towards Unfolded Proteins and ATP-Dependent Activity for Folded Proteins
Authors: Fukui Toshiaki , Eguchi Tomohiro , Atomi Haruyuki , Imanaka Tadayuki
Published: July 2002

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain,Archaea. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon fromThermococcus kodakaraensisKOD1 (LonTk) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA+superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that LonTkwas actually a membrane-bound protein. The recombinant LonTkpossessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70°C, respectively. Unlike the enzyme fromEscherichia coli, we found that LonTkshowed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that LonTkrequired ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, LonTkdegraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in LonTkdifferent from that of its bacterial counterpart.
Amino Acid Substitution within the VP7 Protein of G2 Rotavirus Strains Associated with Failure To Serotype
Authors: Gómara Miren Iturriza , Cubitt David , Desselberger Ulrich , Gray Jim
Published: July 2002

Rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped as G2 by reverse transcription-PCR failed to serotype in enzyme-linked immunosorbent assays using three different G2-specific monoclonal antibodies. The deduced amino acid sequences of the antigenic regions A (amino acids 87 to 101), B (amino acids 142 to 152), and C (amino acids 208 to 221) of VP7 revealed that a substitution at position 96 (Asp→Asn) correlated with the change in ability to serotype these G2 strains.
Detection of Rifampin Resistance in
Mycobacterium tuberculosis
in a Single Tube with Molecular Beacons
Authors: El-Hajj Hiyam H. , Marras Salvatore A. E. , Tyagi Sanjay , Kramer Fred Russell , Alland David
Published: July 2002

Current clinical assays for determining antibiotic susceptibility in
Mycobacterium tuberculosis
require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant
M. tuberculosis
in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene,
rpoB
. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the
rpoB
gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible
M. tuberculosis
is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148
M. tuberculosis
clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.
Case of Catheter Sepsis with
Ralstonia gilardii
in a Child with Acute Lymphoblastic Leukemia
Authors: Wauters Georges , Claeys Geert , Verschraegen Gerda , De Baere Thierry , Vandecruys Els , Van Simaey Leen , De Ganck Catharine , Vaneechoutte Mario
Published: July 2002

Acute lymphoblastic leukemia was diagnosed in a 7-year-old girl. Two months after insertion of a central venous catheter, she developed fever and complained of headache and abdominal pain. Physical examination revealed no focus of infection. A gram-negative nonfermenting bacillus was recurrently cultured from blood. Extensive biochemical testing and 16S ribosomal DNA sequencing led to the identification of
Ralstonia gilardii
.
Comparison of Serogroup W-135 Meningococci Isolated in Sweden during a 23-Year Period and Those Associated with a Recent Hajj Pilgrimage
Authors: Mölling Paula , Bäckman Anders , Olcén Per , Fredlund Hans
Published: July 2002

An outbreak of serogroup W-135 meningococcal disease was reported among pilgrims returning from the annual hajj (pilgrimage to Mecca) in mid-March 2000. Molecular characterization was used to investigate the similarity of the hajj-associated W-135 strains with those isolated in Sweden during a 23-year period (1978 to 2000). The same hajj-associated genosubtype, genosubtype P1.5,2,36b, has been documented in Sweden since 1979, while pulsed-field gel electrophoresis and the sulfadiazine resistance of the W-135 isolates indicated that the outbreak was probably due to a new clone of W-135 meningococci.
Laboratory Diagnosis of Human Neurocysticercosis: Double-Blind Comparison of Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Assay
Authors: Proaño-Narvaez J. V. , Meza-Lucas Antonio , Mata-Ruiz Olga , García-Jerónimo Roberto Carlos , Correa Dolores
Published: July 2002

Neurocysticercosis is a common disease in underdeveloped countries. Its diagnosis is based on clinical, imaging (tomography or magnetic resonance), epidemiological, and laboratory data. Several methods based on the detection of antibodies against cysticerci in cerebrospinal fluid or serum have been tested. Among them, an enzyme-linked immunosorbent assay (ELISA) based on the use of a crude parasite antigen has been used by the laboratory network of cysticercosis in Mexico, which has given support to clinicians for up to 7 years. A
Taenia solium
-specific glycoprotein-based electroimmunotransfer blot (EITB) assay was reported to be highly sensitive and specific for this purpose. In order to compare both techniques, we studied 100 neurocysticercosis patients and 70 neurological noncysticercosis controls and searched for specific antibodies in paired samples of serum and cerebrospinal fluid using both techniques. We found that the EITB assay is more sensitive than the ELISA, especially when serum is being tested. Both techniques are more sensitive in cases with multiple living cysts than in cases with single cysts or calcified lesions. No global differences among cases with parasites located in different parts of the central nervous system were found. In the patients with cysts within the parenchyma, the sensitivity of the EITB assay was higher with serum than with cerebrospinal fluid. The immunodominant bands were found to be the same as those previously reported, i.e., GP-39 to -42, GP-24, and GP-13. Based on these results, we suggest the use of the EITB assay in routine diagnosis of cysticercosis for clinical cases.
Transient Fungemia Caused by an Amphotericin B-Resistant Isolate of
Candida haemulonii
Authors: Rodero Laura , Cuenca-Estrella Manuel , Córdoba Susana , Cahn Pedro , Davel Graciela , Kaufman Sara , Guelfand Liliana , Rodríguez-Tudela Juan L.
Published: July 2002

A bloodstream infection due to
Candida haemulonii
afflicting a patient with fever and a medical history of megaloblastic anemia is reported. The clinical isolate was misidentified by the API 20C and VITEK identification systems. The results of susceptibility tests showed that the MIC of amphotericin B for
C. haemulonii
was 4 μg/ml. Additional susceptibility testing procedures based on the use of antibiotic medium 3 and Iso-Sensitest broth were performed, and killing curves were determined. Two collection strains of
C. haemulonii
were employed as controls. The three isolates exhibited resistance to amphotericin B in vitro regardless of the antifungal susceptibility testing method employed. In addition, the MICs of fluconazole for the three isolates were high. Further studies are needed in order to ascertain whether this species exhibits innate or acquired resistance to amphotericin B and other antifungal agents.
Effect of the Murine Leukemia Virus Extended Packaging Signal on the Rates and Locations of Retroviral Recombination
Authors: Anderson Jeffrey A. , Pathak Vinay K. , Hu Wei-Shau
Published: July 2002

Reverse transcriptase (RT) switches templates frequently during DNA synthesis; the acceptor template can be the same RNA (intramolecular) or the copackaged RNA (intermolecular). Previous results indicated that intramolecular template switching occurred far more frequently than intermolecular template switching. We hypothesized that intermolecular template-switching events (recombination) occurred at a lower efficiency because the copackaged RNA was not accessible to the RT. To test our hypothesis, the murine leukemia virus (MLV) extended packaging signal (Ψ
+
) containing a dimer linkage structure (DLS) was relocated from the 5′ untranslated region (UTR) to between selectable markers, allowing the two viral RNAs to interact closely in this region. It was found that the overall maximum recombination rates of vectors with Ψ
+
in the 5′ UTR or Ψ
+
between selectable markers were not drastically different. However, vectors with Ψ
+
located between selectable markers reached a plateau of recombination rate at a shorter distance. This suggested a limited enhancement of recombination by Ψ
+
. The locations of the recombination events were also examined by using restriction enzyme markers. Recombination occurred in all four regions between the selectable markers; the region containing 5′ Ψ
+
including DLS did not undergo more recombination than expected from the size of the region. These experiments indicated that although the accessibility of the copackaged RNA was important in recombination, other factors existed to limit the number of viruses that were capable of undergoing intermolecular template switching. In addition, recombinants with multiple template switches were observed at a frequency much higher than expected, indicating the presence of high negative interference in the MLV-based system. This extends our observation with the spleen necrosis virus system and suggests that high negative interference may be a common phenomenon in retroviral recombination.
Human Immunodeficiency Virus-Specific Circulating CD8 T Lymphocytes Have Down-Modulated CD3ζ and CD28, Key Signaling Molecules for T-Cell Activation
Authors: Trimble Linda A. , Shankar Premlata , Patterson Mark , Daily Johanna P. , Lieberman Judy
Published: July 2002

Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3ζ, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3ζ down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3ζ-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3ζ−. CD8 T cells with down-modulated CD3ζ also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR+CD62L−). After T-cell activation, CD3ζ-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor α-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3ζ is not reexpressed even after IL-2 exposure.
Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies
Authors: Pfeiffer Julie K. , Georgiadis Millie M. , Telesnitsky Alice
Published: July 2002

Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functional
lacZ
gene generation from vectors in which
lacZ
was disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.
Expression and Characterization of the Borna Disease Virus Polymerase
Authors: Walker Michelle Portlance , Jordan Ingo , Briese Thomas , Fischer Nicole , Lipkin W. Ian
Published: July 2002

Borna disease virus
is the prototype of a new family,
Bornaviridae
, within the order
Mononegavirales
, that is characterized by nuclear transcription, splicing, low level replication, and neurotropism. The products of five open reading frames predicted from the genomic sequence have been confirmed; however, expression of the sixth, corresponding to the putative viral polymerase (L), has not been demonstrated. Here, we describe expression and characterization of a 190-kDa protein proposed to represent L. Expression of this protein from the third transcription unit of the viral genome is dependent on a splicing event that fuses a small upstream open reading frame in frame with the larger downstream continuous open reading frame. The protein is detected by serum antibodies from infected rats and is present in the nucleus, where it colocalizes with the phosphoprotein. L is also shown to be phosphorylated by cellular kinases and to interact with the viral phosphoprotein in coimmunoprecipitation studies. These findings are consistent with the identity of the 190-kDa protein as the viral polymerase and provide insights and describe reagents that will be useful for
Bornavirus
molecular biology and pathobiology.