bluuebunny/crossref_metadata_2025_split huggingface dataset:
Staphylococcus aureusFibronectin Binding Proteins Are Essential for Internalization by Osteoblasts but Do Not Account for Differences in Intracellular Levels of Bacteria
Authors: Ahmed Saddif , Meghji Sajeda , Williams Rachel J. , Henderson Brian , Brock Jeremy H. , Nair Sean P. 
Published: July 2002

ABSTRACTStaphylococcus aureusis a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake ofS. aureusby osteoblasts. AnS. aureusseptic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains ofS. aureusblocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains ofS. aureusare internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.
Priming by DNA Immunization Augments Protective Efficacy ofMycobacterium bovisBacille Calmette-Guerin against Tuberculosis
Authors: Feng Carl G. , Palendira Umaimainthan , Demangel Caroline , Spratt Joanne M. , Malin Adam S. , Britton Warwick J. 
Published: July 2002

ABSTRACTSequential immunization with mycobacterial antigen Ag85B-expressing DNA andMycobacterium bovisbacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting againstMycobacterium tuberculosisinfection. Depletion of the CD8+T cells in the immunized mice impaired protection in their spleens, indicating that this improved efficacy was partially mediated by CD8+T cells.
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

ABSTRACT

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

ABSTRACTBordetella 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

ABSTRACTGram-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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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.