bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipooligosaccharide and Polysaccharide Capsule: Virulence Factors ofNeisseria meningitidisThat Determine Meningococcal Interaction with Human Dendritic Cells
Authors: Unkmeir Alexandra , Kämmerer Ulrike , Stade Anne , Hübner Claudia , Haller Sabine , Kolb-Mäurer Annette , Frosch Matthias , Dietrich Guido 
Published: July 2002

ABSTRACTIn this work we analyzed the roles of meningococcal lipooligosaccharide (LOS) and capsule expression in the interaction ofNeisseria meningitidiswith human dendritic cells (DC). Infection of DC with serogroup B wild-type meningococci induced a strong burst of the proinflammatory cytokines and chemokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. In contrast, a serogroup B mutant strain lacking LOS expression barely led to cytokine induction, demonstrating that meningococcal LOS is the main mediator of the proinflammatory response in human DC. Sialylation of meningococcal LOS did not influence cytokine secretion by DC. However, we found the phagocytosis ofN. meningitidisby human DC to be inhibited by LOS sialylation. In addition, the expression of the meningococcal serogroup A, B, and C capsules dramatically reduced DC adherenceof N. meningitidisand phagocytosis to some extent. Hence, LOS sialylation and capsule expression are independent mechanisms protectingN. meningitidisfrom the phagocytic activity of human DC.
Glucan Is a Component of theMycobacterium tuberculosisSurface That Is Expressed In Vitro and In Vivo
Authors: Schwebach J. Reid , Glatman-Freedman Aharona , Gunther-Cummins Leslie , Dai Zhongdong , Robbins John B. , Schneerson Rachel , Casadevall Arturo 
Published: July 2002

ABSTRACTThe outermost layer ofMycobacterium tuberculosisis composed primarily of two polysaccharides, glucan (GC) and arabinomannan. To analyze the surface polysaccharide composition ofM. tuberculosis, we generated a monoclonal antibody (MAb) that bindsM. tuberculosisGC and is known as MAb 24c5. Immunofluorescence and whole-mount immunoelectron microscopy indicated that GC is on the outermost portion of the bacteria.M. tuberculosisstrains Erdman and CDC 1551 were analyzed for their ability to bind MAb 24c5 after in vitro growth in media with and without the detergent Tween 80. MAb 24c5 bound to Erdman and CDC 1551 at all culture times with only slightly greater apparent affinity after extended culture in the absence of Tween 80, indicating that a stable amount of GC polysaccharide antigen is associated with the cell surface ofM. tuberculosis. An enzyme-linked immunosorbent assay indicated that GC is antigenically similar to glycogen, and the amount of GC antigen increased in the media ofM. tuberculosiscultures grown either with or without the detergent Tween 80. Other nontuberculosis mycobacteria have antigenically similar GCs on their surfaces after in vitro growth. Inoculation of mice with live bacilli but not inoculation with dead bacilli elicited a strong antibody response to GC consistent with production of this antigen in vivo. Our results provide a more comprehensive picture of theM. tuberculosiscell envelope and the conditions that allow expression ofM. tuberculosisGC.
MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions ofSalmonellaLipid A
Authors: Muroi Masashi , Ohnishi Takahiro , Tanamoto Ken-ichi 
Published: July 2002

ABSTRACTSalmonellalipid A is inactive in human macrophages despite being potently active in murine macrophages. We investigated the molecular basis for this species-specific action ofSalmonellalipid A. When murine CD14 (mCD14), mTLR4, and mMD-2 were all expressed in human monocytic THP-1 cells, these cells were capable of responding toSalmonellalipid A. Expressing each of these proteins separately did not impart such responsiveness. Expression of mTLR4 plus mMD-2, but not mCD14 plus mTLR4 or mCD14 plus mMD-2, conferred this responsiveness. In THP-1 cells expressing mCD14, mTLR4, and mMD-2, replacing mCD14 with human CD14 had no effect on responsiveness toSalmonellalipid A or syntheticSalmonella-type lipid A (compound 516). When mTLR4 was replaced with human TLR4, the responses to these lipid A preparations were decreased to half, and the replacement of mMD-2 decreased responsiveness to one-third, although the responses toEscherichia colilipid A or syntheticE. coli-type lipid A (compound 506) were not affected. These results indicate that both TLR4 and MD-2 participate in the species-specific action ofSalmonellalipid A.
Synergic Effects of Mycoplasmal Lipopeptides and Extracellular ATP on Activation of Macrophages
Authors: Into Takeshi , Fujita Mari , Okusawa Tsugumi , Hasebe Akira , Morita Manabu , Shibata Ken-Ichiro 
Published: July 2002

ABSTRACTMycoplasmal lipopeptidesS-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF andS-(2,3-bispalmitoyloxypropyl)-CGNNDESNISFKEK activated a monocytic cell line, THP-1 cells, to produce tumor necrosis factor alpha. The activity of the lipopeptides was augmented by ATP in a dose-dependent manner. In addition, the level of expression of mRNAs for tumor necrosis factor alpha and interleukin-1β, -6, and -8 was also upregulated by the lipopeptides and/or extracellular ATP, but that of interleukin-10 was not. The P2X purinergic receptor antagonists pyridoxal phosphate 6-azophenyl 2′,4′-disulfonic acid and periodate-oxidized ATP suppressed the activity of ATP to augment the activation of THP-1 cells by the lipopeptides, suggesting that P2X receptors play important roles in the activity of ATP. The nuclear factor κB inhibitor dexamethasone also suppressed the activity, suggesting that the activity of ATP is dependent upon the nuclear factor κB. Thus, these results suggest that the interaction of extracellular ATP with the P2X receptors is attributed to the activity of ATP to augment the activation of THP-1 cells by mycoplasmal lipopeptides.
Prephenate Dehydratase from the Aphid Endosymbiont (
Buchnera
) Displays Changes in the Regulatory Domain That Suggest Its Desensitization to Inhibition by Phenylalanine
Authors: Jiménez Nuria , González-Candelas Fernando , Silva Francisco J. 
Published: July 2002

ABSTRACT

Buchnera aphidicola
, the prokaryotic endosymbiont of aphids, complements dietary deficiencies with the synthesis and provision of several essential amino acids. We have cloned and sequenced a region of the genome of
B. aphidicola
isolated from
Acyrthosiphon pisum
which includes the two-domain
aroQ/pheA
gene. This gene encodes the bifunctional chorismate mutase-prephenate dehydratase protein, which plays a central role in
l
-phenylalanine biosynthesis. Two changes involved in the overproduction of this amino acid have been detected. First, the absence of an attenuator region suggests a constitutive expression of this gene. Second, the regulatory domain of the
Buchnera
prephenate dehydratase shows changes in the ESRP sequence, which is involved in the allosteric binding of phenylalanine and is strongly conserved in prephenate dehydratase proteins from practically all known organisms. These changes suggest the desensitization of the enzyme to inhibition by phenylalanine and would permit the bacterial endosymbiont to overproduce phenylalanine.
Interaction between FliE and FlgB, a Proximal Rod Component of the Flagellar Basal Body of
Salmonella
Authors: Minamino Tohru , Yamaguchi Shigeru , Macnab Robert M. 
Published: July 2002

ABSTRACT

FliE is a flagellar basal body protein of
Salmonella
whose detailed location and function have not been established. A mutant allele of
fliE
, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to
flgB
, one of four genes encoding the basal body rod; the
fliE flgB
pseudorevertants were better flagellated and swarmed better than the
fliE
parent, especially when the temperature was reduced from 37 to 30°C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parental
fliE
mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the
fliE
mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.
Cloning, Expression, and Purification of the K5 Capsular Polysaccharide Lyase (KflA) from Coliphage K5A: Evidence for Two Distinct K5 Lyase Enzymes
Authors: Clarke Bradley R. , Esumeh Fred , Roberts Ian S. 
Published: July 2002

ABSTRACT

The
Escherichia coli
K5 capsular polysaccharide [-4)-βGlcA-(1,4)-αGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (
kflA
) was cloned and sequenced. The
kflA
gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of
E. coli
SEBR 3282. There was only limited nucleotide homology between the
kflA
and
elmA
genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that
kflA
was not present on the chromosome of the
E. coli
strains examined. In contrast,
elmA
was present in a subset of
E. coli
strains. Homology was observed between DNA flanking the
kflA
gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF
L
) located 5′ of the endosialidase gene of the
E. coli
K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF
L
in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF
L
is a truncated remnant of KflA. The presence of this truncated
kflA
gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional
kflA
. A (His)
6
-KflA fusion protein was overexpressed in
E. coli
and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.
Reconstitution and Partial Characterization of Phospholipid Flippase Activity from Detergent Extracts of the
Bacillus subtilis
Cell Membrane
Authors: Hrafnsdóttir Sigrún , Menon Anant K. 
Published: July 2002

ABSTRACT

In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer. Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible. However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed. To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of
Bacillus subtilis
into proteoliposomes and measured import of a water-soluble phospholipid analog. We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment. Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A
2
-based assay. Protein-free liposomes were inactive. We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities. Glycerol gradient analysis showed that the majority of the transport activity sedimented at ∼4S, correlating with the presence of specific proteins. Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins. We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids. The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.
Molecular Analysis of Sucrose Metabolism of
Erwinia amylovora
and Influence on Bacterial Virulence
Authors: Bogs Jochen , Geider Klaus 
Published: July 2002

ABSTRACT

Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen
Erwinia amylovora
, a chromosomal fragment which enabled
Escherichia coli
to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the
scr
regulon of
E. amylovora
was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes
scrK
,
scrY
,
scrA
,
scrB
, and
scrR
, had high homology to genes of the
scr
regulons from
Klebsiella pneumoniae
and plasmid pUR400.
scrB
and
scrR
of
E. amylovora
were fused to a histidine tag and to the maltose-binding protein (MalE) of
E. coli
, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a

K
m

of 125 mM. Binding of a MalE-ScrR fusion protein to an
scrYAB
promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the
scr
regulon was studied with an
scrYAB
promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by
E. amylovora
.
Molecular Analyses of a Putative CTXφ Precursor and Evidence for Independent Acquisition of Distinct CTXφs by Toxigenic
Vibrio cholerae
Authors: Boyd E. Fidelma , Heilpern Andrew J. , Waldor Matthew K. 
Published: July 2002

ABSTRACT

The genes encoding cholera toxin (
ctxA
and
ctxB
) are encoded in the genome of CTXφ, a filamentous phage that infects
Vibrio cholerae
. To study the evolutionary history of CTXφ, we examined genome diversity in CTXφs derived from a variety of epidemic and nonepidemic
Vibrio
sp. natural isolates. Among these were three
V. cholerae
strains that contained CTX prophage sequences but not the
ctxA
and
ctxB
genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXφ replicative form, with the exception of missing
ctxAB
. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5′ of
ctxA
, as well as the
ctxAB
promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXφ precursor that lacked
ctxAB
simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTXφs, two CTXφ-encoded genes,
orfU
and
zot
, were sequenced from 13
V. cholerae
and 4
V. mimicus
isolates. Comparative nucleotide sequence analyses revealed that the CTXφs derived from classical and El Tor
V. cholerae
isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on
mdh
sequences). These findings suggest that nontoxigenic precursors of the two
V. cholerae
O1 biotypes independently acquired distinct CTXφs.
Sequence Changes in the Ton Box Region of BtuB Affect Its Transport Activities and Interaction with TonB Protein
Authors: Cadieux Nathalie , Bradbeer Clive , Kadner Robert J. 
Published: July 2002

ABSTRACT

Uptake of cobalamins by the transporter protein BtuB in the outer membrane of
Escherichia coli
requires the proton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.
Sequence of the Genome of the Temperate, Serotype-Converting,
Pseudomonas aeruginosa
Bacteriophage D3
Authors: Kropinski Andrew M. 
Published: July 2002

ABSTRACT

Temperate bacteriophage D3, a member of the virus family
Siphoviridae
, is responsible for serotype conversion in its host,
Pseudomonas aeruginosa
. The complete sequence of the double-stranded DNA genome has been determined. The 56,426 bp contains 90 putative open reading frames (ORFs) and four genes specifying tRNAs. The latter are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The tRNAs may function in the translation of certain highly expressed proteins from this relatively AT-rich genome. D3 proteins which exhibited a high degree of sequence similarity to previously characterized phage proteins included the portal, major head, tail, and tail tape measure proteins, endolysin, integrase, helicase, and NinG. The layout of genes was reminiscent of lambdoid phages, with the exception of the placement of the endolysin gene, which parenthetically also lacked a cognate holin. The greatest sequence similarity was found in the morphogenesis genes to coliphages HK022 and HK97. Among the ORFs was discovered the gene encoding the fucosamine
O
-acetylase, which is in part responsible for the serotype conversion events.
Identification of the
ubiD
Gene on the
Escherichia coli
Chromosome
Authors: Zhang Haitao , Javor George T. 
Published: July 2002

ABSTRACT

The open reading frame at 86.7 min on the
Escherichia coli
chromosome, “
yigC
,” complemented a
ubiD
mutant strain, AN66, indicating that
yigC
is the
ubiD
gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.
VisN and VisR Are Global Regulators of Chemotaxis, Flagellar, and Motility Genes in
Sinorhizobium (Rhizobium) meliloti
Authors: Sourjik Victor , Muschler Paul , Scharf Birgit , Schmitt Rüdiger 
Published: July 2002

ABSTRACT

The known 41 flagellar, chemotaxis, and motility genes of
Sinorhizobium (Rhizobium) meliloti
contained in the “flagellar regulon” are organized as seven operons and six transcription units that map to a contiguous 45-kb chromosomal region. By probing gene expression on Western blots and with
lacZ
fusions, we have identified two master regulatory genes,
visN
and
visR
, contained in one operon. The gene products probably form a heterodimer, VisNR, acting as a global transcription activator of other flagellar genes. The related 27-kDa VisN and VisR proteins are LuxR-type proteins with typical ligand- and DNA-binding domains. The
vis
operon itself is constitutively transcribed; however, to activate flagellar genes, VisNR seemingly requires the binding of a yet-unknown effector. Gene expression in tester strains with known deficiencies revealed a hierarchy of three classes of flagellar genes: class I comprises
visN
and
visR
; class II, controlled by VisNR, comprises flagellar assembly (class IIA) and motor (class IIB) genes; and class III comprises flagellin and chemotaxis genes that require functional class I and class IIA genes for expression. In contrast to their enterobacterial counterparts,
mot
genes belong to class II without exerting control over class III genes. While the general hierarchy of gene expression resembles the enterobacterial scheme, the assignment of
mot
genes to class IIB and the global control by a LuxR-type VisNR activator are new features distinguishing the
S. meliloti
flagellar gene system.