bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Enhanced Th1 Response afterLeishmania amazonensisInfection of C57BL/6 Interleukin-10-Deficient Mice Does Not Lead to Resolution of Infection
Authors: Jones Douglas E. , Ackermann Mark R. , Wille Ulrike , Hunter Christopher A. , Scott Phillip
Published: July 2002

C3H and C57BL/6 mice are resistant toLeishmania majorbut develop chronic lesions with persistent parasite loads when they are infected withLeishmania amazonensis. These lesions develop in the absence of interleukin-4 (IL-4), indicating that susceptibility to this parasite is not a result of development of a Th2 response. Expression of the cytokine IL-10 during infection could account for the lack of IL-12 expression and poor cell-mediated immunity towards the parasite. Therefore, we tested the hypothesis that IL-10 plays a central role in downmodulating the Th1 response afterL. amazonensisinfection. Infection of C57BL/6 IL-10-deficient mice indicated that in the absence of IL-10 there was early enhancement of a Th1 response, which was downregulated during the more chronic stage of infection. In addition, although there were 1- to 2-log reductions in the parasite loads within the lesions, the parasites continued to persist, and they were associated with chronic lesions whose size was similar to that of the control lesions. These experiments indicated thatL. amazonensisresistance to killing in vivo is only partially dependent on expression of host IL-10. However, IL-10-deficient mice had an enhanced delayed-type hypersensitivity response during the chronic phase of infection, indicating that there were Th1 type effector cells in vivo at this late stage of infection. These results indicate that although IL-10 plays a role in limiting the Th1 response during the acute infection phase, other immunomodulatory factors are responsible for limiting the Th1 response during the chronic phase.
Genomic Analysis of the F3031 Brazilian Purpuric Fever Clone of
Haemophilus influenzae
Biogroup Aegyptius by PCR-Based Subtractive Hybridization
Authors: Smoot Laura M. , Franke Deanna D. , McGillivary Glen , Actis Luis A.
Published: July 2002

PCR-based subtractive genome hybridization produced clones harboring inserts present in Brazilian purpuric fever (BPF) prototype strain F3031 but absent in noninvasive
Haemophilus influenzae
biogroup aegyptius isolate F1947. Some of these inserts have no matches in the GenBank database, while others are similar to genes encoding either known or hypothetical proteins. One insert represents a 2.3-kb locus with similarity to a
Thermotoga maritima
hypothetical protein, while another is part of a 7.6-kb locus that contains predicted genes encoding hypothetical, phage-related, and carotovoricin Er-like proteins. The presence of DNA related to these loci is variable among BPF isolates and nontypeable
H. influenzae
strains, while neither of them was detected in strains of types a to f. The data indicate that BPF-causing strain F3031 harbors unique chromosomal regions, most of which appear to be acquired from unrelated microbial sources.
Apoptosis in Acute Shigellosis Is Associated with Increased Production of Fas/Fas Ligand, Perforin, Caspase-1, and Caspase-3 but Reduced Production of Bcl-2 and Interleukin-2
Authors: Raqib Rubhana , Ekberg Caroline , Sharkar Protim , Bardhan Pradip K. , Zychlinsky Arturo , Sansonetti Philippe J. , Andersson Jan
Published: July 2002

Shigella dysenteriaetype 1-induced apoptotic cell death in rectal tissues from patients infected withShigella dysenteriaetype 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P< 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase ofS. dysenteriaetype 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival.
Role of the
dpr
Product in Oxygen Tolerance in
Streptococcus mutans
Authors: Yamamoto Yuji , Higuchi Masako , Poole Leslie B. , Kamio Yoshiyuki
Published: July 2002

We have previously identified and characterized the alkyl hydroperoxide reductase of
Streptococcus mutans
, which consists of two components, Nox-1 and AhpC. Deletion of both
nox-1
and
ahpC
had no effect on the sensitivity of
S. mutans
to cumene hydroperoxide or H
2
O
2
, implying that the existence of another antioxidant system(s) independent of the Nox-1–AhpC system compensates for the deficiency. Here, a new antioxidant gene (
dpr
for Dps-like peroxide resistance gene) was isolated from the
S. mutans
chromosome by its ability to complement an
ahpCF
deletion mutant of
Escherichia coli
with a
tert
-butyl hydroperoxide-hypersensitive phenotype. The
dpr
gene complemented the defect in peroxidase activity caused by the deletion of
nox-1
and
ahpC
in
S. mutans
. Under aerobic conditions, the
dpr
disruption mutant carrying a spectinomycin resistance gene (
dpr
::Spc
r
mutant) grew as well as wild-type
S. mutans
in liquid medium. However, the
dpr
::Spc
r
mutant could not form colonies on an agar plate under air. In addition, neither the
dpr
::Spc
r
ahpC
::Em
r
::
nox-1
triple mutant nor the
dpr
::Spc
r
sod
::Em
r
double mutant was able to grow aerobically in liquid medium. The 20-kDa
dpr
gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of
S. mutans
cells to air. We propose a mechanism by which Dpr confers aerotolerance on
S. mutans.
Mobilization of Chimeric
oriT
Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity
Authors: Fekete Richard A. , Frost Laura S.
Published: July 2002

Cleavage at the F plasmid
nic
site within the origin of transfer (
oriT
) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at
nic
in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1
oriT
regions or various combinations of
nic
, TraY, and TraM binding sites, in addition to the
traM
gene. The efficiency of cleavage at
nic
and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F
traM
mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at
nic
was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of
nic
, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within
oriT
. High-affinity binding of TraY in
cis
to
oriT
allowed detection of cleavage at
nic
but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to
oriT
are preferentially targeted to the transfer apparatus (transferosome).
Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in
Lactococcus lactis
Authors: Melchiorsen Claus Rix , Jokumsen Kirsten Væver , Villadsen John , Johnsen Mads G. , Israelsen Hans , Arnau José
Published: July 2002

The enzyme pyruvate formate-lyase (PFL) from
Lactococcus lactis
was produced in
Escherichia coli
and purified to obtain anti-PFL antibodies that were shown to be specific for
L. lactis
PFL. It was demonstrated that activated
L. lactis
PFL was sensitive to oxygen, as in
E. coli
, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of
L. lactis
, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In
E. coli
, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in
L. lactis
, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in
E. coli
and
L. lactis
.
Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of
metE
and
metH
: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase
Authors: Fritsch Paula S. , Urbanowski Mark L. , Stauffer George V.
Published: July 2002

Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of
metE
and
metH
. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either
metE
only (residues 260, 276, 302, 306, 309, and 322) or
metH
only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for
metE
activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α
2
, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of
metE
, while MetR interacts preferentially with the αCTD on the β-associated α subunit at
metH
. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.
The Overlapping
angB
and
angG
Genes Are Encoded within the
trans
-Acting Factor Region of the Virulence Plasmid in
Vibrio anguillarum
: Essential Role in Siderophore Biosynthesis
Authors: Welch Timothy J. , Chai Sunghee , Crosa Jorge H.
Published: July 2002

Products encoded in the
trans
-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of
Vibrio anguillarum
. Here we identify
angB
, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis. We demonstrate that a 287-amino-acid polypeptide, encoded by
angB
and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2,3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate. Complementation of various
angB
mutations provided evidence that an additional, overlapping gene exists at this locus. This second gene, designated
angG
, also has an essential biosynthetic function. The
angG
gene directs the expression of three polypeptides when overexpressed in
Escherichia coli
, all of which are translated in the same frame as AngB. The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule. Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the
angB
and
angG
genes.
A Thermostable Single-Strand DNase from
Methanococcus jannaschii
Related to the RecJ Recombination and Repair Exonuclease from
Escherichia coli
Authors: Rajman Luis A. , Lovett Susan T.
Published: July 2002

The RecJ protein of
Escherichia coli
plays an important role in a number of DNA repair and recombination pathways. RecJ catalyzes processive degradation of single-stranded DNA in a 5′-to-3′ direction. Sequences highly related to those encoding RecJ can be found in most of the eubacterial genomes sequenced to date. From alignment of these sequences, seven conserved motifs are apparent. At least five of these motifs are shared among a large family of proteins in eubacteria, eukaryotes, and archaea, including the PPX1 polyphosphatase of yeast and
Drosophila
Prune. Archaeal genomes are particularly rich in such sequences, but it has not been clear whether any of the encoded proteins play a functional role similar to that of RecJ exonuclease. We have investigated three such proteins from
Methanococcus jannaschii
with the strongest overall sequence similarity to
E. coli
RecJ. Two of the genes, MJ0977 and MJ0831, partially complement a
recJ
mutant phenotype in
E. coli
. The expression of MJ0977 in
E. coli
resulted in high levels of a thermostable single-stranded DNase activity with properties similar to those of RecJ exonuclease. Despite overall weak sequence similarity between the MJ0977 product and RecJ, these nucleases are likely to have similar biological functions.
Regulation by Overlapping Promoters of the Rate of Synthesis and Deposition into Crystalline Inclusions of
Bacillus thuringiensis
δ-Endotoxins
Authors: Sedlak Mira , Walter Thomas , Aronson Arthur
Published: July 2002

During sporulation,
Bacillus thuringiensis
produces intracellular, crystalline inclusions comprised of a mixture of protoxins active on insect larvae. A major class of these protoxin genes, designated
cry1
, is transcribed from two overlapping promoters (BtI and BtII) utilizing RNA polymerase containing sporulation sigma factors ς
E
and ς
K
, respectively. Fusions of these promoters to
lacZ
were constructed in order to analyze transcription patterns. Mutations within the −10 region of the BtII promoter (within the spacer region of the BtI promoter) which departed from the consensus −10 sequence for either ς
E
or ς
K
resulted in inactivation of transcription from BtII and a fivefold stimulation of transcription from BtI. In contrast, transcription from both promoters was inhibited with a change to the ς
E
consensus. One of the “promoter-up” mutations was fused to the
cry1Ac1
gene, and enhanced transcription was confirmed by Northern blotting. There was an increase in the accumulation of Cry1Ac antigen at early but not later times in sporulation in the mutant. This shift was due to the rapid turnover of much of the excessively accumulated protoxin at the early times as measured by pulse-chase labeling. As a result of the turnover and the inactivation of the BtII promoter, the mutant produced smaller inclusions which contained two- to threefold-less protoxin than inclusions from the wild type. Promoter overlap is a mechanism for modulating protoxin synthesis, thus ensuring the efficient packaging of these protoxins into inclusions.
Global Regulatory Mutations in
csrA
and
rpoS
Cause Severe Central Carbon Stress in
Escherichia coli
in the Presence of Acetate
Authors: Wei Bangdong , Shin Sooan , LaPorte David , Wolfe Alan J. , Romeo Tony
Published: July 2002

The
csrA
gene encodes a small RNA-binding protein, which acts as a global regulator in
Escherichia coli
and other bacteria (T. Romeo, Mol. Microbiol. 29:1321–1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of
csrA
in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of
csrA rpoS
mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of ≥25 mM acetate consisted substantially of glycogen biosynthesis (
glg
) mutants, which were no longer inhibited by acetate. Several classes of
glg
mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that
csrA
positively regulates
acs
(acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the
glg
mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the
csrA rpoS
strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.
A Study of AroP-PheP Chimeric Proteins and Identification of a Residue Involved in Tryptophan Transport
Authors: Cosgriff Angela J. , Brasier Geoff , Pi Jing , Dogovski Con , Sarsero Joseph P. , Pittard A. J.
Published: July 2002

In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins.
Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes
Authors: Šmajs David , Weinstock George M.
Published: July 2002

The 5.2-kb ColJs plasmid of a colicinogenic strain of
Shigella sonnei
(colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the
rep
,
mob
, and
cer
regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (
cja
), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (
cji
) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (
cjl
) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the
cjl
gene was found upstream from
cja
. Moreover, the promoter upstream from
cjl
was similar to promoters described upstream from several colicin activity genes. The
cji
gene was found to be located downstream from
cja
with a transcription polarity opposite to that of the
cjl
and
cja
genes. The
cja
,
cji
, and
cjl
genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus.
Construction of an In Vivo Nonsense Readthrough Assay System and Functional Analysis of Ribosomal Proteins S12, S4, and S5 in
Bacillus subtilis
Authors: Inaoka Takashi , Kasai Koji , Ochi Kozo
Published: July 2002

To investigate the function of ribosomal proteins and translational factors in
Bacillus subtilis
, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like
Escherichia coli
mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (
ram
) mutants from an S12 mutant. These
ram
mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a
ram
phenotype in
B. subtilis
. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.
Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole
Authors: Epple Ulrike D. , Suriapranata Ivet , Eskelinen Eeva-Liisa , Thumm Michael
Published: July 2002

Selective disintegration of membrane-enclosed autophagic bodies is a feature of eukaryotic cells not studied in detail. Using a
Saccharomyces cerevisiae
mutant defective in autophagic-body breakdown, we identified and characterized Aut5p, a glycosylated integral membrane protein. Site-directed mutagenesis demonstrated the relevance of its putative lipase active-site motif for autophagic-body breakdown.
aut5
Δ cells show reduced protein turnover during starvation and are defective in maturation of proaminopeptidase I. Most recently, by means of the latter phenotype, Aut5p was independently identified as Cvt17p. In this study we additionally checked for effects on vacuolar acidification and detected mature vacuolar proteases, both of which are prerequisites for autophagic-body lysis. Furthermore, biologically active hemagglutinin-tagged Aut5p (Aut5-Ha) localizes to the endoplasmic reticulum (nuclear envelope) and is targeted to the vacuolar lumen independent of autophagy. In
pep4
Δ cells immunogold electron microscopy located Aut5-Ha at ∼50-nm-diameter intravacuolar vesicles. Characteristic missorting in
vps
class E and
fab1
Δ cells, which affects the multivesicular body (MVB) pathway, suggests vacuolar targeting of Aut5-Ha similar to that of the MVB pathway. In agreement with localization of Aut5-Ha at intravacuolar vesicles in
pep4
Δ cells and the lack of vacuolar Aut5-Ha in wild-type cells, our pulse-chase experiments clearly indicated that Aut5-Ha degradation with 50 to 70 min of half-life is dependent on vacuolar proteinase A.
CheR- and CheB-Dependent Chemosensory Adaptation System of
Rhodobacter sphaeroides
Authors: Martin Angela C. , Wadhams George H. , Shah Deepan S. H. , Porter Steven L. , Mantotta Jeevani C. , Craig Tim J. , Verdult Peter H. , Jones Helen , Armitage Judith P.
Published: July 2002

Rhodobacter sphaeroides
has multiple homologues of most of the
Escherichia coli
chemotaxis genes, organized in three major operons and other, unlinked, loci. These include
cheA

1

and
cheR

1

(
che
Op
1
) and
cheA

2

,

cheR
2

, and

cheB
1

(
che
Op
2
). In-frame deletions of these
cheR
and
cheB
homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR
2
and CheB
1
were essential for normal chemotaxis, whereas CheR
1
was not.
cheR

2

and
cheB

1

, but not
cheR

1

, were also able to complement the equivalent
E. coli
mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in
R. sphaeroides
. In
E. coli,
CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any
R. sphaeroides
chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR
2
either interacts with the NWETF motif of
E. coli
methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that
R. sphaeroides
has an adaptation system that is different from that of
Bacillus subtilis
and
E. coli,
with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA
2
, but not CheA
1
, is able to phosphorylate CheB
1
, suggesting that signaling through CheA
1
cannot initiate feedback receptor adaptation via CheB
1
-P.
Complementation of Cold Shock Proteins by Translation Initiation Factor IF1 In Vivo
Authors: Weber Michael H. W. , Beckering Carsten L. , Marahiel Mohamed A.
Published: July 2002

The cold shock response in both
Escherichia coli
and
Bacillus subtilis
is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For
B. subtilis
it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the
B. subtilis cspB cspC
double deletion mutant revealed that removal of these
csp
genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from
E. coli
in a
B. subtilis cspB cspC
double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.
Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL from
Azotobacter vinelandii
Authors: Money Tracy , Barrett Jason , Dixon Ray , Austin Sara
Published: July 2002

The enhancer binding protein NIFA and the sensor protein NIFL from
Azotobacter vinelandii
comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.
Quinone Profiles of
Thermoplasma acidophilum
HO-62
Authors: Shimada Haruo , Shida Yasuo , Nemoto Naoki , Oshima Tairo , Yamagishi Akihiko
Published: July 2002

Quinones of
Thermoplasma acidophilum
HO-62 were analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. Menaquinone, methionaquinone, and 2-
trans
and 2-
cis
forms of thermoplasmaquinone were identified. The relative amount of thermoplasmaquinone increased under anaerobic conditions, and those of menaquinone and methionaquinone increased under aerobic conditions.
OmpC Is the Receptor for Gifsy-1 and Gifsy-2 Bacteriophages of
Salmonella
Authors: Ho Theresa D. , Slauch James M.
Published: July 2002

Mutations in the
Salmonella enterica
serovar Typhimurium
ompC
gene conferred resistance to Gifsy-1 and Gifsy-2 bacteriophages. Selection for complementing plasmids yielded clones of
ompC
. Introduction of an
ompC
clone into
Escherichia coli
conferred the ability to adsorb Gifsy phage. These data show that OmpC is the receptor for Gifsy-1 and Gifsy-2 phages.
In Vitro Transposition System for Efficient Generation of Random Mutants of
Campylobacter jejuni
Authors: Colegio Oscar R. , Griffin Thomas J. , Grindley Nigel D. F. , Galán Jorge E.
Published: July 2002

Campylobacter jejuni
is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the
Staphylococcus aureus
transposable element Tn
552
that allows the efficient generation of insertion mutants of
C. jejuni
. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of
C. jejuni
. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.
Growth Phase Variation in Cell and Nucleoid Morphology in a
Bacillus subtilis recA
Mutant
Authors: Sciochetti Stephen A. , Blakely Garry W. , Piggot Patrick J.
Published: July 2002

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth,
Bacillus subtilis recA
cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for
addB
and
recF
mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (∼15%) of abnormally long cells and nucleoids developed in
B. subtilis recA
mutants. In addition,
recA
mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.
Examination of the
Borrelia burgdorferi
Transcriptome in
Ixodes scapularis
during Feeding
Authors: Narasimhan Sukanya , Santiago Felix , Koski Raymond A. , Brei Brandon , Anderson John F. , Fish Durland , Fikrig Erol
Published: July 2002

Borrelia burgdorferi
gene expression within the guts of engorging
Ixodes scapularis
ticks was examined by use of differential immunoscreening and differential expression with a customized amplified library. Fourteen chromosomal genes involved in energy metabolism, substrate transport, and signal transduction and 10 (4 chromosomal and 6 plasmid) genes encoding putative lipoproteins and periplasmic proteins were preferentially expressed in engorging ticks. These data demonstrate a new approach to the global analysis of
B. burgdorferi
genes that are preferentially expressed within the vector during feeding.
Analysis of the Heat Shock Response of
Neisseria meningitidis
with cDNA- and Oligonucleotide-Based DNA Microarrays
Authors: Guckenberger Matthias , Kurz Sebastian , Aepinus Christian , Theiss Stephanie , Haller Sabine , Leimbach Thomas , Panzner Ursula , Weber Jacqueline , Paul Hubert , Unkmeir Alexandra , Frosch Matthias , Dietrich Guido
Published: July 2002

Oligonucleotide- and cDNA-based microarrays comprising a subset of
Neisseria meningitidis
genes were assessed for study of the meningococcal heat shock response and found to be highly suitable for transcriptional profiling of
N. meningitidis
. Employing oligonucleotide arrays encompassing the entire genome of
N. meningitidis
, we analyzed the meningococcal heat shock response on a global scale and identified 55 heat shock-deregulated open reading frames (34 induced and 21 repressed).
Characterization of
AFMP1
: a Novel Target for Serodiagnosis of Aspergillosis
Authors: Yuen Kwok-Yung , Chan Che-Man , Chan King-Man , Woo Patrick C. Y. , Che Xiao-Yan , Leung Andy S. P. , Cao Liang
Published: July 2002

We cloned the
AFMP1
gene, which encodes the first antigenic cell wall galactomannoprotein in
Aspergillus fumigatus. AFMP1
codes for a protein, Afmp1p, of 284 amino acid residues, with a few sequence features that are present in Mp1p, the antigenic cell wall mannoprotein in
Penicillium marneffei
that we described previously, as well as several other cell wall proteins of
Saccharomyces cerevisiae
and
Candida albicans
. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidyl inositol attachment signal sequence. Specific anti-Afmp1p antibody was generated with recombinant Afmp1p protein purified from
Escherichia coli
to allow further characterization of Afmp1p. Afmp1p has a high affinity for
Galanthus nivalis
agglutinin, a characteristic indicative of a mannoprotein. Furthermore, it was recognized by a rat monoclonal antibody against the galactofuran side chain of galactomannan, indicating that it is a galactomannoprotein. Ultrastructural analysis by immunogold staining indicated that Afmp1p is present in the cell walls of the hyphae and conidia of
A. fumigatus
. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to
A. fumigatus
develop a specific antibody response against Afmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.
Characterization of Enteropathogenic and Enteroaggregative
Escherichia coli
Isolated from Diarrheal Outbreaks
Authors: Yatsuyanagi Jun , Saito Shioko , Sato Hiroyasu , Miyajima Yoshimichi , Amano Ken-Ichi , Enomoto Katsuhiko
Published: July 2002

Virulence characteristics of diarrheal outbreak-associated
Escherichia coli
O55:NM, O126:NM, and O111:NM were examined. The
E. coli
O55:NM strains were atypical enteropathogenic
E. coli
(EPEC), while the
E. coli
O126:NM and O111:NM strains should be classified as enteroaggregative
E. coli
(EAggEC). The contributions of EPEC and EAggEC to the human disease burden in Japan might be significantly greater than is currently appreciated.
Comparison of Three Commercially Available Peptide-Based Immunoglobulin G (IgG) and IgA Assays to Microimmunofluorescence Assay for Detection of
Chlamydia trachomatis
Antibodies
Authors: Morré Servaas A. , Munk Christian , Persson Kenneth , Krüger-Kjaer Susanne , van Dijk Rogier , Meijer Chris J. L. M. , van den Brule Adriaan J. C.
Published: July 2002

Three commercially available, peptide-based enzyme-linked immunosorbent assay (ELISA) systems (Chlamydia trachomatis IgG and IgA EIA [CT-EIA; Labsystems OY, Helsinki, Finland], SeroCT IgG and IgA [SeroCT; Savyon Diagnostics Ltd., Ashdod, Israel], and Chlamydia trachomatis IgG and IgA pELISA [CT pELISA; Medac, Wedel, Germany]) were evaluated for the detection of serum immunoglobulin G (IgG) and IgA antibodies specific for
Chlamydia trachomatis
and compared to the “gold standard” assay, the microimmunofluorescence (MIF) assay. Serological responses were analyzed in 149 women aged 20 to 30 years. Cervical swabs obtained from these women were examined for
C. trachomatis
by PCR, and 43 were found to be positive. The overall seroprevalence rates detected by CT-EIA, SeroCT, CT pELISA, and the MIF assay were 42, 42, 35, and 39%, respectively, for IgG and 7, 7, 3, and 7%, respectively, for IgA. The IgG seroprevalence rates for the PCR-positive women were two to three times higher than those for the PCR-negative women, i.e., 72 versus 29%, 72 versus 29%, 47 versus 26%, and 74 versus 25% for CT-EIA, SeroCT, CT pELISA, and the MIF assay, respectively. After discrepancy analysis, the sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the IgG assays; for CT-EIA they were 84.7, 98.6, 98.4, and 86.7%, respectively; for CT pELISA they were 71.4, 97.3, 96.2, and 78.3%, respectively; for SeroCT they were 84.7, 98.6, 98.4, and 86.3%, respectively; and for the MIF assay they were 79.2, 83.1, 98.3, and 83.1%, respectively. In conclusion, these peptide-based ELISA systems for the serological detection of
C. trachomatis
infection performed as well as the MIF assay. Since these tests are less time-consuming, less expensive, and easier to perform than the MIF assay, they might be useful in the serodiagnosis of chlamydial infection.
Rapid and Sensitive Detection of Physical Status of Human Papillomavirus Type 16 DNA by Quantitative Real-Time PCR
Authors: Nagao Shoji , Yoshinouchi Mitsuo , Miyagi Yasunari , Hongo Atsushi , Kodama Junichi , Itoh Sachio , Kudo Takafumi
Published: July 2002

A rapid quantitative real-time PCR method was employed to quantify the copy number of E2 and E6 genes for analysis of the physical status of human papillomavirus type 16 (HPV-16) DNA. Significant differences with respect to both copy numbers were found when more than 40% of HPV-16 DNA was integrated with disruption of the E2 gene in an experimental model. The physical status of HPV-16 DNA in 50 clinical samples was exclusively episomal in 21 cases (42%), concomitant in 14 cases (28%), and integrated in 15 cases (30%). The prevalence of integrated and/or concomitant forms of HPV-16 DNA increased with progression of cervical disease. Four of 11 cervical intraepithelial neoplasia involved integrated forms of HPV-16 DNA partially or exclusively. This rapid, sensitive technique is useful in the analysis of the physical status of HPV DNA.
New Real-Time PCR Able To Detect in a Single Tube Multiple Rifampin Resistance Mutations and High-Level Isoniazid Resistance Mutations in
Mycobacterium tuberculosis
Authors: García de Viedma Darío , del Sol Díaz Infantes Maria , Lasala Fátima , Chaves Fernando , Alcalá Luis , Bouza Emilio
Published: July 2002

The emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for
Mycobacterium tuberculosis
is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)- and isoniazid (INH)-resistant
M. tuberculosis
strains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the
rpoB
core region in all the RIF-resistant (RIF
r
) strains. Forty-six percent of the INH-resistant (INH
r
) strains showed a mutation in
katG
codon 315, and most of these were associated with high MICs. Eighteen of the RIF
r
, INH
r
, and multidrug-resistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.
Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus
Authors: Saijo Masayuki , Qing Tang , Niikura Masahiro , Maeda Akihiko , Ikegami Tetsuro , Prehaud Christophe , Kurane Ichiro , Morikawa Shigeru
Published: July 2002

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni
2+
column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections.
Discrimination between Cases of Duodenal Ulcer and Gastritis on the Basis of Putative Virulence Factors of
Helicobacter pylori
Authors: Yamaoka Yoshio , Souchek Julianne , Odenbreit Stefan , Haas Rainer , Arnqvist Anna , Borén Thomas , Kodama Tadashi , Osato Michael S. , Gutierrez Oscar , Kim Jong G. , Graham David Y.
Published: July 2002

The BabA,
cagA
, and
vacA
statuses of 827
Helicobacter pylori
isolates were used in logistic regression models to discriminate duodenal ulcer from gastritis. Only BabA was a candidate for a universal virulence factor, but the low
c
statistic value (0.581) indicates that none of these factors were helpful in predicting the clinical presentation.
Disseminated Herpes Simplex Virus and Varicella Zoster Virus Coinfection in a Patient Taking Thalidomide for Relapsed Multiple Myeloma
Authors: Curley Matthew J. , Hussein Shakir A. , Hassoun Paul M.
Published: July 2002

Disseminated herpes simplex virus (HSV) and varicella zoster virus (VZV) have been reported individually in immunosuppressed adults. We present a case of coinfection with disseminated HSV and VZV infection in a patient taking thalidomide for relapsed multiple myeloma. This is the first report of opportunistic infection associated with thalidomide.
Complement Depletion Facilitates the Infection of Multiple Brain Tumors by an Intravascular, Replication-Conditional Herpes Simplex Virus Mutant
Authors: Ikeda Keiro , Wakimoto Hiroaki , Ichikawa Tomotsugu , Jhung Sarah , Hochberg Fred H. , Louis David N. , Chiocca E. Antonio
Published: July 2002

Intravascular routes of administration can provide a means to target gene- and virus-based therapies to multiple tumor foci located within an organ, such as the brain. However, we demonstrate here that rodent plasma inhibits cell transduction by replication-conditional (oncolytic) herpes simplex viruses (HSV), replication-defective HSV, and adenovirus vectors. In vitro depletion of complement with mild heat treatment or in vivo depletion by treatment of athymic rats with cobra venom factor (CVF) partially reverses this effect. Without CVF, inhibition of cell infection by HSV is observed at plasma dilution as high as 1:32, while plasma from CVF-treated animals displays anti-HSV activity at lower dilutions (1:8). When applied to the therapy of intracerebral brain tumors, in vivo complement depletion facilitates the initial infection (assayed at the 2-day time point) by an intra-arterial replication-conditional HSV of tumor cells, located within three separate and distinct human glioma masses. However, at the 4-day time point, no propagation of HSV from initially infected tumor cells could be observed. Previously, we have shown that the immunosuppressive agent, cyclophosphamide (CPA), facilitates the in vivo propagation of an oncolytic HSV, delivered intravascularly, within infected multiple intracerebral masses, by inhibition of both innate and elicited anti-HSV neutralizing antibody response (K. Ikeda et al., Nat. Med. 5:881–889, 1999). In this study, we thus show that the addition of CPA to the CVF treatment results in a significant increase in viral propagation within infected tumors, measured at the 4-day time period. The concerted action of CVF and CPA significantly increases the life span of athymic rodents harboring three separate and large glioma xenografts after treatment with intravascular, oncolytic HSV. Southern analysis of viral genomes analyzed by PCR reveals the presence of the oncolytic virus in the brains, livers, spleens, kidneys, and intestine of treated animals, although none of these tissues displays evidence of HSV-mediated gene expression. In light of clinical trials of oncolytic HSV for malignant brain tumors, these findings suggest that antitumor efficacy may be limited by the host innate and elicited humoral responses.