bluuebunny/crossref_metadata_2025 huggingface dataset:
Toxoplasma gondii-Infected Human Myeloid Dendritic Cells Induce T-Lymphocyte Dysfunction and Contact-Dependent Apoptosis
Authors: Wei Shuang , Marches Florentina , Borvak Jozef , Zou Weiping , Channon Jacqueline , White Michael , Radke Jay , Cesbron-Delauw Marie-France , Curiel Tyler J.
Published: July 2002

Dendritic cells ignite adaptive immunity by priming naïve T lymphocytes. Human monocyte-derived dendritic cells (MDDCs) infected withToxoplasma gondiiinduce T-lymphocyte gamma interferon production and may thus activateT. gondii-specific immunity. However, we now demonstrate thatT. gondii-infected MDDCs are poor at activating T lymphocytes and are unable to induce specific cytotoxic T lymphocytes. On the other hand, MDDCs acquiring nonviableT. gondiiantigens directly, or indirectly through captured apoptotic or necrotic cell bodies, induce potent T-lymphocyte activation. T lymphocytes exposed to infected MDDCs are significantly impaired in upregulation of CD69 and CD28, are refractory to activation, and die through contact-dependent apoptosis mediated by an as-yet-unidentified mechanism not requiring Fas, tumor necrosis factor-related apoptosis-inducing ligand, leukocyte function antigen 1, intercellular adhesion molecule 1, tumor necrosis factor alpha, interleukin 10, alpha interferon, gamma interferon, prostaglandins, or reactive nitrogen intermediates. Bystander T lymphocytes that were neither infected nor apoptotic were refractory to activation, suggesting global dysfunction. Immunosuppression and T-lymphocyte unresponsiveness and apoptosis are typical of acuteT. gondiiinfection. Our data suggest that infected dendritic cells contribute to these processes. On the other hand, host cells infected withT. gondiiare resistant to multiple inducers of apoptosis. Thus, regulation of host cell and bystander cell apoptosis by viableT. gondiimay be significant components of a strategy to evade immunity and enhance intracellular parasite survival.
Upregulation of Thymosin β-10 byMycobacterium bovisInfection of Bovine Macrophages Is Associated with Apoptosis
Authors: Gutiérrez-Pabello José A. , McMurray David N. , Adams L. Garry
Published: July 2002

Bovine macrophages underwent apoptosis as a result of infection with aMycobacterium bovisfield strain. Macrophages infected with a multiplicity of infection (MOI) of 25:1 developed chromatin condensation and DNA fragmentation at 4 h and 8 h, respectively, whereas changes in chromatin condensation induced by MOIs of 10:1 and 1:1 required more time and had a reduced number of apoptotic cells. Not only infected macrophages underwent apoptosis, but also uninfected bystander macrophages became apoptotic. Increased differential expression of thymosin β-10 was identified inM. bovis-infected bovine macrophages by differential display reverse transcriptase PCR. Phagocytosis of latex beads had no effect on the expression of thymosin β-10, whereas bacterial suspensions upregulated thymosin β-10 expression, suggesting thatM. bovisor mycobacterial products are essential in the process. Heat-inactivatedM. bovisinduced a slight increase in thymosin β-10 mRNA, whereas live virulent and attenuatedM. bovisorganisms increased the gene expression almost twofold. A mouse macrophage cell line (RAW 264.7) overexpressing the bovine thymosin β-10 transgene had spontaneous apoptosis at a higher rate (66.5%) than parental cells (4.7%) or RAW cells harboring the empty vector (22.8%). The apoptotic rates of the overexpressing cells were significantly higher when compared with both the empty vector transfected (P< 0.01) and parental cells (P< 0.001). Our evidence suggests that upregulation of thymosin β-10 inM. bovis-infected macrophages is linked with increased cell death due to apoptosis.
Functional Substitution of the TibC Protein of Enterotoxigenic
Escherichia coli
Strains for the Autotransporter Adhesin Heptosyltransferase of the AIDA System
Authors: Moormann Corinna , Benz Inga , Schmidt M. Alexander
Published: July 2002

The plasmid-encoded AIDA (adhesin involved in diffuse adherence) autotransporter protein derived from diffuse-adhering clinical
Escherichia coli
isolate 2787 and the TibA (enterotoxigenic invasion locus B) protein encoded by the chromosomal
tib
locus of enterotoxigenic
E. coli
(ETEC) strain H10407 are posttranslationally modified by carbohydrate substituents. Analysis of the AIDA-I adhesin showed that the modification involved heptose residues. AIDA-I is modified by the heptosyltransferase activity of the product of the
aah
gene, which is located directly upstream of adhesin-encoding gene
aidA
. The carbohydrate modification of the TibA adhesin/invasin is mediated by the TibC protein but has not been elucidated. Based on the sequence similarities between TibC and AAH (autotransporter adhesin heptosyltransferase) and between the TibA and the AIDA proteins we hypothesized that the AIDA system and the Tib system encoded by the
tib
locus are structurally and functionally related. Here we show that (i) TibC proteins derived from different ETEC strains appear to be highly conserved, (ii) recombinant TibC proteins can substitute for the AAH heptosyltransferase in introducing the heptosyl modification to AIDA-I, (iii) this modification is functional in restoring the adhesive function of AIDA-I, (iv) a single amino acid substitution at position 358 completely abolishes this activity, and (v) antibodies directed at the functionally active AIDA-I recognize a protein resembling modified TibA in ETEC strains. In summary, we conclude that, like AAH, TibC represents an example of a novel class of heptosyltransferases specifically transferring heptose residues onto multiple sites of a protein backbone. A potential consensus sequence for the modification site is suggested.
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

In 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

The 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

Salmonellalipid 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

Mycoplasmal 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

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

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

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

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

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

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

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

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

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

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.
Two Extracytoplasmic Function Sigma Subunits, ς
E
and ς
FecI
, of
Escherichia coli
: Promoter Selectivity and Intracellular Levels
Authors: Maeda Hiroto , Jishage Miki , Nomura Tasuku , Fujita Nobuyuki , Ishihama Akira
Published: July 2002

The promoter selectivity of two extracytoplasmic function (ECF) subfamily ς subunits, ς
E
(ς
24
) and ς
FecI
(ς
18
), of
Escherichia coli
RNA polymerase was analyzed by using an in vitro transcription system and various promoters. The Eς
E
holoenzyme recognized only the known cognate promoters,
rpoE
P2,
rpo
HP3, and
degP
, and the Eς
FecI
recognized only one known cognate promoter,
fecA
. The strict promoter recognition properties of ς
E
and ς
FecI
are similar to those of other minor ς subunits. Transcription by Eς
E
and Eς
FecI
was enhanced by high concentrations of glutamate, as in the case of other minor ς subunits. The optimum temperature for transcription by Eς
FecI
was low, around 25°C, apparently in agreement with the high rate of iron sequestration by
E. coli
at low temperatures. By quantitative Western blot analysis, the intracellular levels of ς
E
and ς
FecI
in the uninduced steady-state culture of
E. coli
W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per μg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major ς
70
subunit.
Regulation of the
cnr
Cobalt and Nickel Resistance Determinant of
Ralstonia eutropha
(
Alcaligenes eutrophus
) CH34
Authors: Tibazarwa C. , Wuertz S. , Mergeay M. , Wyns L. , van der Lelie D.
Published: July 2002

The linked resistance to nickel and cobalt of
Ralstonia eutropha
-like strain CH34 (
Alcaligenes eutrophus
CH34) is encoded by the
cnr
operon, which is localized on the megaplasmid pMOL28. The regulatory genes
cnrYXH
have been cloned, overexpressed, and purified in
Escherichia coli
. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in
E. coli
. His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of
E. coli
core RNA polymerase enzyme, could retard at least two different promoter DNA targets,
cnrYp
and
cnrHp
, localized within the
cnrYXH
locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL
M91650
description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of
cnrYXH
and the
luxCDABE
, luciferase reporter genes. Nickel and cobalt specifically induced the
cnrYXH-luxCDABE
fusion at optimal concentrations of 0.3 mM Ni
2+
and 2.0 mM Co
2+
in a noncomplexing medium for metals. The two promoter regions P
Y
(upstream
cnrY
) and P
H
(upstream
cnrH
) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the
cnr
operon. The
cnrHp
promoter was responsible for full transcription of the
cnrCBA
structural resistance genes, while the
cnrYp
promoter was necessary to obtain metal-inducible transcription from the
cnrHp
promoter. The zinc resistance phenotype (ZinB) of a spontaneous
cnr
mutant strain, AE963, was investigated and could be attributed to an insertion of IS
1087
, a member of the IS
2
family of insertion elements, within the
cnrY
gene.
RegA, Iron, and Growth Phase Regulate Expression of the
Pseudomonas aeruginosa tol-oprL
Gene Cluster
Authors: Duan Kangmin , Lafontaine Eric R. , Majumdar Sonali , Sokol Pamela A.
Published: July 2002

The
tol-oprL
region in
Pseudomonas aeruginosa
appears to be involved in pyocin uptake and required for cell viability. The complete nucleotide sequences of the
tolQRA
and
oprL
genes as well as the incomplete sequences of
tolB
and
orf2
have been previously reported. In addition, the sequence of a
P. aeruginosa
iron-regulated gene (
pig6
) has been described and found to share homology with an open reading frame located upstream of the
Escherichia coli tolQRA
genes (U. A. Ochsner and M. L. Vasil, Proc. Natl. Acad. Sci. USA 93:4409–4414, 1996). In this study, we cloned the remainder of the
P. aeruginosa tol-oprL
gene cluster and determined its nucleotide sequence. This cluster was found to consist of seven genes in the order
orf1 tolQ tolR tolA tolB oprL orf2
. Transcriptional analysis of this gene cluster was performed by detecting the presence of mRNAs spanning adjacent genes as well as by using a promoterless
lacZ
reporter gene fused to each of the seven genes contained in the
tol-oprL
locus. The results show that there are three major transcriptional units or operons in this region,
orf1-tolQRA
,
tolB
, and
oprL-orf2
, in contrast to the
E. coli tol-pal
region, where there are only two operons,
orf1-tolQRA
and
tolB-pal-orf2
. Analysis of gene expression indicated that the
tol-oprL
genes of
P. aeruginosa
are both iron and growth phase modulated. The first operon,
orf1-tolQRA
, is iron regulated throughout growth, but iron-regulated expression of
tolB
and
oprL
fusions occurs only in late log phase. The expression of the three operons was significantly less repressed by iron in
fur
mutants than in the wild-type strain, suggesting the involvement of Fur in the iron regulation of all three operons. RegA is a positive yet nonessential regulator of
tol-oprL
expression.
Domain Structure, Oligomeric State, and Mutational Analysis of PpsR, the
Rhodobacter sphaeroides
Repressor of Photosystem Gene Expression
Authors: Gomelsky Mark , Horne Irene M. , Lee Hye-Joo , Pemberton John M. , McEwan Alastair G. , Kaplan Samuel
Published: July 2002

The transcription factor PpsR from the facultative photoheterotroph
Rhodobacter sphaeroides
is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in
ppsR
. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action.
Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase
Authors: Lassy Rachel A. L. , Miller Charles G.
Published: July 2002

Salmonella entericaserovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) inEscherichia coliand have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in theArchaeaor in any of the other eukaryotes for which genome sequences are available.
Characterization of
Escherichia coli
Type 1 Pilus Mutants with Altered Binding Specificities
Authors: Harris Sandra L. , Spears Patricia A. , Havell Edward A. , Hamrick Terri S. , Horton John R. , Orndorff Paul E.
Published: July 2002

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in
fimH
, the chromosomal gene that encodes the adhesin protein (FimH) of
Escherichia coli
type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.
Insertional Mutagenesis in the
n
-Alkane-Assimilating Yeast
Yarrowia lipolytica
: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization
Authors: Mauersberger Stephan , Wang Hui-Jie , Gaillardin Claude , Barth Gerold , Nicaud Jean-Marc
Published: July 2002

Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a
zeta-URA3
mutagenesis cassette (MTC) into the genome of a
zeta
-free and
ura3
deletion-containing strain of
Yarrowia lipolytica
. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura
+
transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk
−
) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk
−
mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc). Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integrated MTC were sequenced to identify the disrupted genes. Sequence analysis indicated that the MTC was integrated in the
LEU1
locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (
PEX14
) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in
Y. lipolytica
.
Analysis of Catabolite Control Protein A-Dependent Repression in
Staphylococcus xylosus
by a Genomic Reporter Gene System
Authors: Jankovic Ivana , Egeter Oliver , Brückner Reinhold
Published: July 2002

A single-copy reporter system for
Staphylococcus xylosus
has been developed, that uses a promoterless version of the endogenous β-galactosidase gene
lacH
as a reporter gene and that allows integration of promoters cloned in front of
lacH
into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of
S. xylosus
promoters by the catabolite control protein CcpA. To test if
lacH
is a suitable reporter gene, β-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the
malRA
maltose utilization operon promoter and autoregulation of the
ccpA
promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (
cre
s), from promoter regions of several
S. xylosus
genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P

veg
II

. For that purpose,
cre
sequences were placed at position +3 or +4 within the transcribed region of P

veg
II

. Measurements of β-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of
ccpA
completely abolished glucose-dependent regulation. Therefore, the tested
cre
s functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in
S. xylosus
was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in
S. xylosus
. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.
Clonal Population Structure of
Pseudomonas stutzeri
, a Species with Exceptional Genetic Diversity
Authors: Rius Núria , Fusté M. Carme , Guasp Caterina , Lalucat Jorge , Lorén José G.
Published: July 2002

Genetic diversity and genetic relationships among 42
Pseudomonas stutzeri
strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this
P. stutzeri
sample exhibited the highest mean genetic diversity (
H
= 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (

I
A

) for the
P. stutzeri
strains analyzed was 1.10. The

I
A

values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of
P. stutzeri
is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium.
Cloning, Sequencing, and Characterization of the Iturin A Operon
Authors: Tsuge Kenji , Akiyama Takanori , Shoda Makoto
Published: July 2002

Bacillus subtilis
RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames,
ituD, ituA, ituB
, and
ituC
. The
ituD
gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene,
ituA
, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene,
ituB
, and the fourth gene,
ituC
, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by
B. subtilis
ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are
d
-Ser→
l
-Asn, while in iturin A these amino acids are inverted (i.e.,
d
-Asn→
l
-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that
ituD, ituA
, and
ituB
have high levels of homology to the counterpart genes
fenF
(79%),
mycA
(79%), and
mycB
(79%), respectively. Although the overall level of homology of the amino acid sequences encoded by
ituC
and
mycC
, the counterpart of
ituC
, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of
xynD
of
B. subtilis
168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of
xynD
imply that horizontal transfer of this operon occurred. When the promoter was replaced by the
repU
promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.