bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogenicity of a 26-Valent Group A Streptococcal Vaccine
Authors: Hu Mary C. , Walls Michael A. , Stroop Steven D. , Reddish Mark A. , Beall Bernard , Dale James B.
Published: July 2002

A multivalent vaccine containing amino-terminal M protein fragments from 26 different serotypes of group A streptococci was constructed by recombinant techniques. The vaccine consisted of four different recombinant proteins that were formulated with alum to contain 400 μg of protein per dose. Rabbits were immunized via the intramuscular route at 0, 4, and 16 weeks. Immune sera were assayed for the presence of type-specific antibodies against the individual recombinant M peptides by enzyme-linked immunosorbent assay and for opsonic antibodies by in vitro opsonization tests and indirect bactericidal tests. The 26-valent vaccine was highly immunogenic and elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. The immune sera were broadly opsonic and were bactericidal against the majority of the 26 different serotypes. Importantly, none of the immune sera cross-reacted with human tissues. Our results indicate that type-specific, protective M protein epitopes can be incorporated into complex, multivalent vaccines designed to elicit broadly protective opsonic antibodies in the absence of tissue-cross-reactive antibodies.
Alpha/Beta Interferon Impairs the Ability of Human Macrophages To Control Growth ofMycobacterium bovisBCG
Authors: Bouchonnet Francine , Boechat Neio , Bonay Marcel , Hance Allan J.
Published: July 2002

Administration of alpha/beta interferon (IFN-α/β) to mice infected withMycobacterium tuberculosishas been shown to increase mycobacterial growth. Because IFN-α/β has direct pleiotropic effects on the differentiation and functional activities of macrophages, we evaluated the effect of IFN-α/β on mycobacterial growth in human monocytes/macrophages in vitro. Monocytes cultured at optimal cell density could control the growth ofM. bovisBCG, as assessed both by measurement of luciferase activity expressed by a mycobacterial reporter strain and by counting of CFU. In contrast, unrestrained mycobacterial growth was observed when monocytes were treated with alpha interferon (IFN-α) 3 days prior to or concomitant with infection. This striking loss of mycobacteriostatic activity was observed with IFN-α and IFN-β and was induced in both freshly isolated monocytes and culture-derived macrophages. Pretreatment of monocytes with IFN-α modified cellular morphology and reduced viability following culture, but neither was observed for culture-derived macrophages, indicating that the effects of IFN-α on mycobacteriostatic activity and cell differentiation and death could be dissociated. These results are compatible with the possibility that the secretion of IFN-α/β could directly promote mycobacterial growth in patients harboring these organisms.
Plasmodium vivaxPromiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens
Authors: Caro-Aguilar Ivette , Rodríguez Alexandra , Calvo-Calle J. Mauricio , Guzmán Fanny , De la Vega Patricia , Elkin Patarroyo Manuel , Galinski Mary R. , Moreno Alberto
Published: July 2002

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 ofPlasmodium vivax(PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previousP. vivaxinfections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with thePlasmodium falciparumimmunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restrictedP. falciparumT-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognizeP. vivaxmerozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion ofP. falciparumsporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.
Purified Malaria Pigment (Hemozoin) Enhances Dendritic Cell Maturation and Modulates the Isotype of Antibodies Induced by a DNA Vaccine
Authors: Coban Cevayir , Ishii Ken J. , Sullivan David J. , Kumar Nirbhay
Published: July 2002

Hemozoin (malaria pigment) has been implicated in the modulation of immune responses during malaria infection. This study was designed to evaluate the effect of purified hemozoin on the in vitro activation of myeloid dendritic cells. Our study also revealed that in addition to enhancing the maturation of dendritic cells, hemozoin also greatly promotes immunoglobulin G2a antibody responses when coadministered with a DNA vaccine plasmid encoding Pfs25, aPlasmodium falciparumtransmission-blocking antigen.
In
Saccharomyces cerevisiae
, Expression of Arginine Catabolic Genes
CAR1
and
CAR2
in Response to Exogenous Nitrogen Availability Is Mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) Complex
Authors: Messenguy Francine , Vierendeels Fabienne , Scherens Bart , Dubois Evelyne
Published: July 2002

The products of three genes named
CARGRI
,
CARGRII
, and
CARGRIII
were shown to repress the expression of
CAR1
and
CAR2
genes, involved in arginine catabolism.
CARGRI
is identical to
UME6
and encodes a regulator of early meiotic genes. In this work we identify
CARGRII
as
SIN3
and
CARGRIII
as
RPD3
. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress
CAR1
expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (
CAR1
) and ornithine transaminase (
CAR2
), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of
CAR1
and
CAR2
, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the
UME6
gene impairs cell growth more strongly than the deletion of the
SIN3
or
RPD3
gene, especially in the Σ1278b background.
Transcriptional Analysis of Major Heat Shock Genes of
Helicobacter pylori
Authors: Homuth Georg , Domm Stephanie , Kleiner Diethelm , Schumann Wolfgang
Published: July 2002

The transcriptional organization and heat inducibility of the major heat shock genes
hrcA
,
dnaK
,
dnaJ
,
groEL
, and
htpG
were analyzed on the transcriptional level in
Helicobacter pylori
strain 69A. The strongly heat-induced
dnaK
operon was found to be tricistronic, consisting of the genes
hrcA
,
grpE
, and
dnaK
. The
dnaJ
gene specified one monocistronic mRNA which was also heat inducible. The genes
groES
and
groEL
were transcribed as one strongly heat-inducible bicistronic mRNA which exhibited exactly the same induction kinetic as the
dnaK
operon. Surprisingly, transcription of the monocistronic
htpG
gene was switched off after heat shock. The data presented are discussed with regard to the different mechanisms regulating expression of heat shock genes in
H. pylori
Metabolic Instability of
Escherichia coli
Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis
Authors: Chang Ying-Ying , Eichel Johannes , Cronan John E.
Published: July 2002

Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In
Escherichia coli
, the onset of CFA synthesis results from increased transcription of
cfa
, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the activity quickly declines to the basal level. We report that the loss of CFA activity is due to proteolytic degradation dependent on expression of the heat shock regulon. CFA synthase degradation is unaffected by mutations in the
lon
,
clpP
, and
groEL
genes or by depletion of the intracellular ATP pools. It seems likely that CFA synthase is the target of an unidentified energy-independent heat shock regulon protease. This seems to be the first example of heat shock-dependent degradation of a normal biosynthetic enzyme.
Analysis of Nucleoid Morphology during Germination and Outgrowth of Spores ofBacillusSpecies
Authors: Ragkousi Katerina , Cowan Ann E. , Ross Margery A. , Setlow Peter
Published: July 2002

After a few minutes of germination, nucleoids in the great majority of spores ofBacillus subtilisandBacillus megateriumwere ring shaped. The major spore DNA binding proteins, the α/β-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did theB. megateriumhomolog of the majorB. subtilischromosomal protein HBsu. The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of α/β-type SASP. As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact. This change took place after degradation of most of the spores' pool of major α/β-type SASP and was delayed when α/β-type SASP degradation was delayed. Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells.
Action of the Thiamine Antagonist Bacimethrin on Thiamine Biosynthesis
Authors: Zilles Julie L. , Croal Laura R. , Downs Diana M.
Published: July 2002

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of
Salmonella enterica
serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the
thi
operon or specific lesions in
thiD
resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the
thiD
mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.
FSY1
, a Novel Gene Encoding a Specific Fructose/H
+
Symporter in the Type Strain of
Saccharomyces carlsbergensis
Authors: Gonçalves Paula , Rodrigues de Sousa Helena , Spencer-Martins Isabel
Published: July 2002

A novel gene,
FSY1
, encoding a permease involved in active fructose uptake by a proton symport mechanism in the type strain of
Saccharomyces carlsbergensis
has been isolated. Fsy1p is only distantly related to the Hxt proteins that mediate facilitated diffusion of glucose and fructose in
Saccharomyces cerevisiae
and related species.
Expression of
cbsA
Encoding the Collagen-Binding S-Protein of
Lactobacillus crispatus
JCM5810 in
Lactobacillus casei
ATCC 393
T
Authors: Martı́nez Beatriz , Sillanpää Jouko , Smit Egbert , Korhonen Timo K. , Pouwels Peter H.
Published: July 2002

The
cbsA
gene encoding the collagen-binding S-layer protein of
Lactobacillus crispatus
JCM5810 was expressed in
L. casei
ATCC 393
T
. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of
L. casei
, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.
Cloning and Characterization of Sialidases with 2-6′ and 2-3′ Sialyl Lactose Specificity from
Pasteurella multocida
Authors: Mizan Shaikh , Henk Adam , Stallings Amy , Maier Marie , Lee Margie D.
Published: July 2002

Pasteurella multocida
is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of
P. multocida
produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene,
nanH
, from a fowl cholera isolate of
P. multocida
. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of
nanH
resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3′ sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from
nanH
in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3′ and 2-6′ sialyl lactose, while NanH demonstrated activity only on 2-3′ sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8′ sialyl lactose). Recombinant
E. coli
containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of
P. multocida
to colonize and persist on vertebrate mucosal surfaces.
Pleiotropic Effect of Protein P6 on the Viral Cycle of Bacteriophage φ29
Authors: Camacho Ana , Salas Margarita
Published: July 2002

The product of bacteriophage φ29 early gene 6, protein p6, is a double-stranded-DNA binding protein and one of the more abundant proteins during viral infection. We have studied the role of protein p6 in vivo through the infection of suppressor and nonsuppressor
Bacillus subtilis
strains with a phage carrying a nonsense mutation in gene 6, sus6(626). In the absence of functional protein p6, the two major processes of the viral cycle, transcription and DNA replication, were affected. Viral DNA synthesis was practically abolished, and early transcription was remarkably delayed and, in addition, underregulated at late times of the infection. The amount of protein p6 synthesized after infection with mutant phage sus6(626) under suppressor conditions was sixfold lower than that produced after wild-type infection. Nonetheless, phage production was as high as that obtained after wild-type infection. These results indicate that p6 is synthesized in amounts higher than those needed for most of its functions. However, the concentration of protein p6 appeared to be important for repression of the early promoter C2.
Cloning and Characterization of the
Flavobacterium johnsoniae
Gliding-Motility Genes
gldB
and
gldC
Authors: Hunnicutt David W. , McBride Mark J.
Published: July 2002

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of mutants of the gliding bacterium
Flavobacterium johnsoniae
(
Cytophaga johnsonae
) with defects in gliding motility have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nongliding mutant of
F. johnsoniae
(UW102-99) with a library of wild-type DNA by using the shuttle cosmid pCP26. The complementing plasmid (pCP200) contained an insert of 26 kb and restored gliding motility to 4 of 50 independently isolated nongliding mutants. A 1.9-kb fragment which encompassed two genes,
gldB
and
gldC
, complemented all four mutants. An insertion mutation in
gldB
was polar on
gldC
, suggesting that the two genes form an operon. Disruption of the chromosomal copy of
gldB
in wild-type
F. johnsoniae
UW101 eliminated gliding motility. Introduction of the
gldBC
operon, or
gldB
alone, restored motility.
gldB
appears to be essential for
F. johnsoniae
gliding motility. It codes for a membrane protein that does not exhibit strong sequence similarity to other proteins in the databases.
gldC
is not absolutely required for gliding motility, but cells that do not produce GldC form colonies that spread less well than those of the wild type. GldC is a soluble protein and has weak sequence similarity to the fungal lectin AOL.
Identification of a Chitin-Binding Protein Secreted by
Pseudomonas aeruginosa
Authors: Folders Jindra , Tommassen Jan , van Loon Leendert C. , Bitter Wilbert
Published: July 2002

One of the major proteins secreted by
Pseudomonas aeruginosa
is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263–270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of
Streptomyces olivaceoviridis
and CBP21 of
Serratia marcescens
and to the cellulose-binding protein p40 of
Streptomyces halstedii
. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of
Vibrio harveyi
. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of
P. aeruginosa
, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).
Partitioning of the Linear Chromosome during Sporulation of
Streptomyces coelicolor
A3(2) Involves an
oriC
-Linked
parAB
Locus
Authors: Kim Hyun-Jin , Calcutt Michael J. , Schmidt Francis J. , Chater Keith F.
Published: July 2002

Candidate partitioning genes (
parA
and
parB
) for the linear chromosome of
Streptomyces coelicolor
were identified by DNA sequencing in a series of seven genes located between
rnpA
and
trxA
near the chromosomal replication origin. The most likely translation start point of
parB
overlapped the
parA
stop codon, suggestive of coregulation, and transcription analysis suggested that the two genes formed an operon. Deletion of part of
parB
had no effect on the growth or appearance of colonies but caused a deficiency in DNA partitioning during the multiple septation events involved in converting aerial hyphae into long chains of spores. At least 13% of spore compartments failed to inherit the normal DNA allocation. The same phenotype was obtained with a deletion removing a segment of DNA from both
parA
and
parB
. Reinforcing the idea of a special role for the
par
locus during sporulation, the stronger of two
parAB
promoters was greatly upregulated at about the time when sporulation septation was maximal in colonies. Three copies of a 14-bp inverted repeat (GTTTCACGTGAAAC) were found in or near the
parAB
genes, and at least 12 more identical copies were identified within 100 kb of
oriC
from the growing genome sequence database. Only one perfect copy of the 14-bp sequence was present in approximately 5 Mb of sequence available from the rest of the genome. The 14-bp sequence was similar to sequences identified as binding sites for Spo0J, a ParB homologue from
Bacillus subtilis
believed to be important for DNA partitioning (D. C.-H. Lin and A. D. Grossman, Cell 92:675–685, 1998). One of these sites encompassed the transcription start point of the stronger
parA
promoter.
Penicillin-Binding Protein-Related Factor A Is Required for Proper Chromosome Segregation in
Bacillus subtilis
Authors: Pedersen Lotte B. , Setlow Peter
Published: July 2002

Previous work has shown that the
ponA
gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with
prfA
(PBP-related factor A) (also called
recU
), which encodes a putative 206-residue basic protein (pI = 10.1) with no significant sequence homology to proteins with known functions. Inactivation of
prfA
results in cells that grow slower and vary significantly in length relative to wild-type cells. We now show that
prfA
mutant cells have a defect in chromosome segregation resulting in the production of ∼0.9 to 3% anucleate cells in
prfA
cultures grown at 30 or 37°C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in
smc
and
spoOJ
mutant cells. In addition, overexpression of
prfA
was found to be toxic for and cause nucleoid condensation in
Escherichia coli.
Structural and Functional Analysis of the Phosphonoacetate Hydrolase (
phnA
) Gene Region in
Pseudomonas fluorescens
23F
Authors: Kulakova Anna N. , Kulakov Leonid A. , Akulenko Natalya V. , Ksenzenko Vladimir N. , Hamilton John T. G. , Quinn John P.
Published: July 2002

The
Pseudomonas fluorescens
23F phosphonoacetate hydrolase gene (
phnA
) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (
phnA
) indicated the presence of five open reading frames (ORFs). These include one (
phnR
) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (
phnB
) that lies immediately downstream of
phnA
and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the
phnAB
and
phnR
operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5′ ends. Transcripts of
phnAB
were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of
phnR
were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the
phnA
region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either
Pseudomonas putida
or
Escherichia coli
cells.
Red Fluorescent Protein (DsRed) as a Reporter in
Saccharomyces cerevisiae
Authors: Rodrigues Fernando , van Hemert Martijn , Steensma H. Yde , Côrte-Real Manuela , Leão Cecı́la
Published: July 2002

We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for
Saccharomyces cerevisiae
. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.
Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in
Pseudomonas aeruginosa
Authors: Köhler Thilo , van Delden Christian , Curty Lasta Kocjancic , Hamzehpour Mehri Michea , Pechere Jean-Claude
Published: July 2002

Intrinsic and acquired antibiotic resistance of the nosocomial pathogen
Pseudomonas aeruginosa
is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that
nfxC
type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the
las
and
rhl
quorum-sensing systems of
P. aeruginosa
. In agreement with these observations are the decreased transcription of the elastase gene
lasB
and the rhamnosyltransferase genes
rhlAB
measured in
nfxC
type mutants. Expression of the
lasR
and
rhlR
regulator genes was not affected in the
nfxC
type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene
rhlI
was reduced by 50% in the
nfxC
type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the
nfxC
type mutant. Transcription of an
rhlAB
-
lacZ
fusion could be partially restored by the addition of synthetic C4-HSL and
Pseudomonas
quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.
The
djlA
Gene Acts Synergistically with
dnaJ
in Promoting
Escherichia coli
Growth
Authors: Genevaux Pierre , Schwager Françoise , Georgopoulos Costa , Kelley William L.
Published: July 2002

The DnaK chaperone of
Escherichia coli
is known to interact with the J domains of DnaJ, CbpA, and DjlA. By constructing multiple mutants, we found that the
djlA
gene was essential for bacterial growth above 37°C in the absence of
dnaJ
. This essentiality depended upon the J domain of DjlA but not upon the normal membrane location of DjlA.
Genetic Locus Required for Antigenic Maturation of
Rhizobium etli
CE3 Lipopolysaccharide
Authors: Duelli Dominik M. , Tobin Andrea , Box Jodie M. , Kolli V. S. Kumar , Carlson Russell W. , Noel K. Dale
Published: July 2002

Rhizobium etli
modifies lipopolysaccharide (LPS) structure in response to environmental signals, such as low pH and anthocyanins. These LPS modifications result in the loss of reactivity with certain monoclonal antibodies. The same antibodies fail to recognize previously isolated
R. etli
mutant strain CE367, even in the absence of such environmental cues. Chemical analysis of the LPS in strain CE367 demonstrated that it lacked the terminal sugar of the wild-type O antigen, 2,3,4-tri-
O
-methylfucose. A 3-kb stretch of DNA, designated as
lpe3
, restored wild-type antigenicity when transferred into CE367. From the sequence of this DNA, five open reading frames were postulated. Site-directed mutagenesis and complementation analysis suggested that the genes were organized in at least two transcriptional units, both of which were required for the production of LPS reactive with the diagnostic antibodies. Growth in anthocyanins or at low pH did not alter the specific expression of
gusA
from the transposon insertion of mutant CE367, nor did the presence of multiple copies of
lpe3
situated behind a strong, constitutive promoter prevent epitope changes induced by these environmental cues. Mutations of the
lpe
genes did not prevent normal nodule development on
Phaseolus vulgaris
and had very little effect on the occupation of nodules in competition with the wild-type strain.
Global Transcriptional Response of
Bacillus subtilis
to Heat Shock
Authors: Helmann John D. , Wu Ming Fang Winston , Kobel Phil A. , Gamo Francisco-Javier , Wilson Michael , Morshedi Maud M. , Navre Marc , Paddon Chris
Published: July 2002

In response to heat stress,
Bacillus subtilis
activates the transcription of well over 100 different genes. Many of these genes are members of a general stress response regulon controlled by the secondary sigma factor, ς
B
, while others are under control of the HrcA or CtsR heat shock regulators. We have used DNA microarrays to monitor the global transcriptional response to heat shock. We find strong induction of known ς
B
-dependent genes with a characteristic rapid induction followed by a return to near prestimulus levels. The HrcA and CtsR regulons are also induced, but with somewhat slower kinetics. Analysis of DNA sequences proximal to newly identified heat-induced genes leads us to propose ∼70 additional members of the ς
B
regulon. We have also identified numerous heat-induced genes that are not members of known heat shock regulons. Notably, we observe very strong induction of arginine biosynthesis and transport operons. Induction of several genes was confirmed by quantitative reverse transcriptase PCR. In addition, the transcriptional responses measured by microarray hybridization compare favorably with the numerous previous studies of heat shock in this organism. Since many different conditions elicit both specific and general stress responses, knowledge of the heat-induced general stress response reported here will be helpful for interpreting future microarray studies of other stress responses.
Cytoplasmic Filament-Deficient Mutant of
Treponema denticola
Has Pleiotropic Defects
Authors: Izard Jacques , Samsonoff William A. , Limberger Ronald J.
Published: July 2002

In
Treponema denticola
, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of
cfpA
and a nonpolar erythromycin resistance cassette (
ermF
and
ermAM
) inserted near the beginning of the gene. The plasmid was electroporated into
T. denticola
, and double-crossover recombinants which had the chromosomal copy of
cfpA
insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in
Treponema
.
Role of CspC and CspE in Regulation of Expression of RpoS and UspA, the Stress Response Proteins in
Escherichia coli
Authors: Phadtare Sangita , Inouye Masayori
Published: July 2002

Nine homologous proteins, CspA to CspI, constitute the CspA family of
Escherichia coli
. Recent studies are aimed at elucidating the individual cellular functions of these proteins. Two members of this family, CspC and CspE, are constitutively produced at 37°C. In the present study, these two proteins were evaluated for their cellular role(s). The expression of three stress proteins, OsmY, Dps, and UspA, is significantly affected by the overexpression and deletion of CspC and CspE. RpoS is a regulatory element for
osmY
and
dps
. Further analysis showed a larger amount and greater stability of the
rpoS
mRNA as well as a higher level of RpoS itself with the overexpression of CspC and CspE. This suggests that CspC and CspE upregulate the expression of OsmY and Dps by regulating the expression of RpoS itself. Indeed, this upregulation is lost in the Δ
rpoS
strain. Other RpoS-controlled proteins such as ProP and KatG, are also upregulated by the overexpression of CspC. The present study suggests that CspC and CspE are the important elements involved in the regulation of the expression of RpoS, a global stress response regulator, and UspA, a protein responding to numerous stresses. In the light of these observations, it seems plausible that CspC and CspE function as regulatory elements for the expression of stress proteins in the complex stress response network of
E. coli
.
Quantitation of the Capacity of the Secretion Apparatus and Requirement for PrsA in Growth and Secretion of α-Amylase in
Bacillus subtilis
Authors: Vitikainen Marika , Pummi Tiina , Airaksinen Ulla , Wahlström Eva , Wu Hongyan , Sarvas Matti , Kontinen Vesa P.
Published: July 2002

Regulated expression of AmyQ α-amylase of
Bacillus amyloliquefaciens
was used to examine the capacity of the protein secretion apparatus of
B. subtilis
. One
B. subtilis
cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 × 10
4
molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of
prsA
and
amyQ
expression levels. Significantly, even when
amyQ
was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of
B. subtilis
. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death.
EvgA of the Two-Component Signal Transduction System Modulates Production of the YhiUV Multidrug Transporter in
Escherichia coli
Authors: Nishino Kunihiko , Yamaguchi Akihito
Published: July 2002

Overexpression of the EvgA regulator of the two-component signal transduction system was previously found to modulate multidrug resistance of
Escherichia coli
by increasing efflux of drugs (K. Nishino and A. Yamaguchi, J. Bacteriol. 183:1455-1458, 2001). Here we present data showing that EvgA contributes to multidrug resistance through increased expression of the multidrug transporter
yhiUV
gene.
Population Heterogeneity of
Salmonella enterica
Serotype Typhimurium Resulting from Phase Variation of the
lpf
Operon In Vitro and In Vivo
Authors: Kingsley Robert A. , Weening Eric H. , Keestra A. Marijke , Bäumler Andreas J.
Published: July 2002

The
lpf
fimbrial operon oscillates between phase ON and phase OFF expression states, thereby generating heterogeneity within
S. enterica
serotype Typhimurium populations with regard to expression of long polar fimbrial antigens. To determine whether the proportion of
lpf
phase variants changes with growth conditions, the
lpf
phase ON content of cultures was determined after in vitro and in vivo passage. After passage in Luria-Bertani (LB) broth for 120 generations, 96% of cells in a serotype Typhimurium culture carried the
lpf
operon in the phase ON expression state, regardless of the phase ON/OFF ratio in the inoculum. In contrast, a culture passaged on LB agar plates for 500 generations contained approximately 2%
lpf
phase ON cells. Differences in the
lpf
phase ON content of cultures passaged in broth and on plates were not caused by an outgrowth of
lpf
phase ON or
lpf
phase OFF cells, since deletion of
lpf
biosynthesis genes did not alter the phase ON/OFF ratio attained after passage. Instead, growth in LB broth resulted in a eightfold increase in the phase OFF-to-ON transition frequency and a decrease of the
lpf
phase ON-to-OFF transition frequency by a factor of 150 compared to growth on LB agar plates. After infection of naïve CBA/J mice with an
lpf
phase ON culture of serotype Typhimurium, the proportion of
lpf
phase ON cells continuously decreased over time, regardless of whether the strain carried intact fimbrial biosynthesis genes. These data suggest that elaboration of fimbriae does not have a major influence on the population heterogeneity produced by phase variation of the
lpf
operon in naïve mice.
Helicobacter typhlonius
sp. nov., a Novel Murine Urease-Negative
Helicobacter
Species
Authors: Franklin Craig L. , Gorelick Peter L. , Riley Lela K. , Dewhirst Floyd E. , Livingston Robert S. , Ward Jerrold M. , Beckwith Catherine S. , Fox James G.
Published: July 2002

Over the past decade, several
Helicobacter
species have been isolated from rodents. With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized
Helicobacter
species also colonize rodents. In this report, we describe a novel urease-negative helicobacter,
Helicobacter typhlonius
sp. nov., which was isolated from colonies of laboratory mice independently by two laboratories. Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections.
H. typhlonius
is genetically most closely related to
H. hepaticus
. Like
H. hepaticus
, it is a spiral bacterium with bipolar sheathed flagella. However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from
H. hepaticus
. Notably,
H. typhlonius
does not produce urease or H
2
S nor does it hydrolize indoxyl-acetate. Compared to other
Helicobacter
species that commonly colonize rodents,
H. typhlonius
was found to be less prevalent than
H. hepaticus
and
H. rodentium
but as prevalent as
H. bilis. H. typhlonius
joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.
Cellular Fatty Acid Composition of
Lautropia mirabilis
Authors: Daneshvar Maryam I. , Douglas Michael P. , Weyant Robbin S.
Published: July 2002

Ten strains of
Lautropia mirabilis
(ATCC 51599
T
and nine phenotypically similar clinical isolates) were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups. The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by gas-liquid chromatography. CFA profiles were generated by using a commerical software package (MIDI, Newark, Del.). All strains tested had an identical CFA profile characterized by major amounts of 16:1ω7c (41%) and 16:0 (44%); smaller amounts (1 to 4%) of 3-OH-10:0, 12:0, 14:0, 15:0, and 18:1 ω7c; trace amounts (<1%) of 10:0, 18:2 and 18:0; and no cyclopropane acids. This profile was similar to the CFA profiles of
Acidovorax delafieldii, Comamonas terrigena
, and strains of an unclassified Centers for Disease Control group designated weak oxidizer group 1. CFA analysis, when supplemented by phenotypic characterization, is useful for the identification of
L. mirabilis
isolates.
Antimicrobial Growth Promoter Ban and Resistance to Macrolides and Vancomycin in Enterococci from Pigs
Authors: Boerlin P. , Wissing A. , Aarestrup F. M. , Frey J. , Nicolet J.
Published: July 2002

Ninety-six enterococcus isolates from fecal samples of pigs receiving tylosin as an antimicrobial growth promoter and 59 isolates obtained in the same farms 5 to 6 months after the ban of antimicrobial growth promoters in Switzerland were tested for susceptibility to nine antimicrobial agents. A clear decrease in resistance to macrolides, lincosamides, and tetracycline was visible after the ban. Vancomycin-resistant
Enterococcus faecium
belonged to the same clonal lineage as vancomycin-resistant isolates previously isolated from Danish pigs.
Detection and Identification of Eight
Trichinella
Genotypes by Reverse Line Blot Hybridization
Authors: Rombout Y. B. , Bosch S. , Van Der Giessen J. W. B.
Published: July 2002

A reverse line blot (RLB) assay was developed to identify different
Trichinella
genotypes. The RLB assay accomplishes detection and specific identification of the different
Trichinella
genotypes and relies on hybridization of the amplified 5S ribosomal DNA intergenic spacer regions to specific, membrane-bound oligonucleotide probes. After one single amplification, we were able to detect and genetically identify six sibling species, i.e.,
T. spiralis
,
T. britovi
,
T. nativa
,
T. murrelli
,
T. nelsoni
, and
T. pseudospiralis
, and two additional
Trichinella
genotypes, T6 and T8. Twenty-four
Trichinella
strains of different genotypes were unequivocally identified evaluated using one simple PCR-based assay based on single larvae. This assay allows the specific identification of
Trichinella
species without the need to passage larvae in laboratory animals.
Development of an Immunoassay for Rapid Detection of Ganglioside GM
1
Mimicry in
Campylobacter jejuni
Strains
Authors: Prendergast Martina M. , Kosunen Timo U. , Moran Anthony P.
Published: July 2002

Mimicry of peripheral nerve gangliosides by
Campylobacter jejuni
lipopolysaccharides (LPSs) has been proposed to induce cross-reacting antiganglioside antibodies in Guillain-Barré syndrome (GBS). Because current methods for LPS characterization are labor-intensive and inhibit the screening of large numbers of strains, a rapid GM
1
epitope screening assay was developed. Biomass from two agar plates of confluent growth yielded sufficient LPS using a novel phenol-water and ether extraction procedure. Extracts of LPS were reacted with cholera toxin (GM
1
ligand), peanut agglutinin (Galβ1→3GalNAc ligand), and anti-GM
1
antibodies. After the assay was validated, 12 of 59 (20%)
C. jejuni
serostrains, including four serotypes that have not previously been associated with GBS, reacted with two or more anti-GM
1
ganglioside reagents. Subsequently, LPS extracts from 5 of 7 (71%)
C. jejuni
isolates and 2 of 3 (67%)
C. jejuni
culture collection strains bore GM
1
structures. Overall, the assay system was reliable, efficient, and reproducible and may be adapted for large-scale epidemiological studies.
Diagnosis and Monitoring of Murine Histoplasmosis by a Nested PCR Assay
Authors: Bialek Ralf , Fischer Jörg , Feucht Antje , Najvar Laura K. , Dietz Klaus , Knobloch Jürgen , Graybill John R.
Published: July 2002

A newly developed nested PCR assay was applied to murine models of histoplasmosis. ICR and BALB/c mice were intravenously infected with
Histoplasma capsulatum
and sacrificed up to 29 days later. Samples of blood, spleen, and lung homogenates were cultured and examined by the PCR assay. In the ICR mouse model, 265 of 319 organ samples showed concordant results. With 7 samples, the culture was positive and the PCR assay was negative whereas a positive PCR but a negative culture were obtained with 47 samples (
P
< 0.0001 according to McNemar's test). Organ homogenates and blood samples of either spontaneously cured or treated BALB/c mice were PCR negative. The nested PCR assay performs excellently in the monitoring of spontaneously and treatment-cured murine histoplasmosis. It limits the infection risks of the laboratory staff and might be of diagnostic value for humans.
Disseminated
Mycobacterium lentiflavum
Infection in a Human Immunodeficiency Virus-Infected Patient
Authors: Niobe S. Ngo , Bebear C. M. , Clerc M. , Pellegrin J.-L. , Bebear C. , Maugein J.
Published: July 2002

We report the first case of
Mycobacterium lentiflavum
disseminated infection in a human immunodeficiency virus-infected patient. Conventional identification procedures failed to identify the mycobacterial strain, but sequencing of the 16S rRNA gene led to the species identification. Furthermore, we describe here the analysis of the 16S-23S rRNA internal transcribed spacer sequence of
M. lentiflavum
.
Strategy for Diagnosis of Congenital Toxoplasmosis: Evaluation of Methods Comparing Mothers and Newborns and Standard Methods for Postnatal Detection of Immunoglobulin G, M, and A Antibodies
Authors: Pinon J. M. , Dumon H. , Chemla C. , Franck J. , Petersen E. , Lebech M. , Zufferey J. , Bessieres M.-H. , Marty P. , Holliman R. , Johnson J. , Luyasu V. , Lecolier B. , Guy E. , Joynson D. H. M. , Decoster A. , Enders G. , Pelloux H. , Candolfi E.
Published: July 2002

In a study involving 14 laboratories supported by the European Community Biomed 2 program, we evaluated immunologic methods for the postnatal diagnosis of congenital toxoplasmosis (CT). Among babies born to mothers who seroconverted to positivity for toxoplasmosis during pregnancy, we analyzed 55 babies with CT on the basis of persistent anti-
Toxoplasma
immunoglobulin G (IgG) at 1 year of life and 50 control babies without anti-
Toxoplasma
IgG at 1 year of life in the absence of curative treatment with pyrimethamine-sulfonamides. We tested in-house methods such as the enzyme-linked immunofiltration assay (ELIFA) or Immunoblotting (IB) for the detection of IgG or IgM; these methods allowed comparison of the immunologic profiles of the mothers and the infants. We compared ELIFA and IB with a commercial enzyme immunoassay (EIA) or in-house immunosorbent agglutination assay (ISAGA) for the detection of IgM or IgA. The performances of combinations of methods were also assessed. A cumulative sensitivity of 98% during a 1-year follow-up was obtained with the ELIFA plus ISAGA combination. Only one case of CT was missed by the ELIFA plus ISAGA combination, whereas three cases were missed by the IB plus ISAGA combination, even though 48% of patients with CT were treated with pyrimethamine-sulfonamides, which are known to inhibit antibody neosynthesis. A similar performance was obtained with either ELIFA or IB in combination with EIA. The difference in performance between ELIFA plus ISAGA and IB plus ISAGA was not statistically significant (
P
= 0.31), and we conclude that both combinations of tests can be used for the diagnosis of CT in newborns.
Suitability of a Rapid Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus in Ghana, West Africa
Authors: Aidoo S. , Ampofo W. K. , Brandful J. A. M. , Nuvor S. V. , Ansah J. K. , Nii-Trebi N. , Barnor J. S. , Apeagyei F. , Sata T. , Ofori-Adjei D. , Ishikawa K.
Published: July 2002

In West African countries such as Ghana, efficient human immunodeficiency virus (HIV) testing is a priority in the fight against AIDS. A new immunochromatographic rapid test, Determine HIV-1/2 (Abbott Diagnostics, North Chicago, Ill.), that detects antibodies against HIV type 1 (HIV-1) and/or HIV-2 was evaluated using Ghanaian blood samples. Two hundred four serum and/or plasma specimens were tested. HIV screening was done by a particle agglutination test and confirmed by a Western blot (WB) test as the “gold standard.” The results revealed 125 HIV-seropositive AIDS patients, 75 HIV-seronegative healthy individuals, and 4 individuals for whom the HIV-1 result was indeterminate. The results obtained by the Determine HIV-1/2 assay and Diagnostic HIV SPOT (Genelabs), which is currently widely used in many districts in Ghana, were compared with those of the WB test, excluding the four HIV-1-indeterminate samples. The sensitivity of the Determine HIV-1/2 assay was 100%, compared with 98.0% for the HIV SPOT assay. The specificity was 100% for both tests. Determine HIV-1/2 is a single-step assay and was found to be rapid and easy to perform without any special equipment. It was highly sensitive and specific. The kit can be applied without electricity and water supplies, making it suitable for the detection of HIV antibodies especially in the rural areas of Ghana, West Africa.
Identification ofEnterococcus, Streptococcus, andStaphylococcusby Multivariate Analysis of Proton Magnetic Resonance Spectroscopic Data from Plate Cultures
Authors: Bourne Roger , Himmelreich Uwe , Sharma Ansuiya , Mountford Carolyn , Sorrell Tania
Published: July 2002

A new fingerprinting technique with the potential for rapid identification of bacteria was developed by combining proton magnetic resonance spectroscopy (1H MRS) with multivariate statistical analysis. This resulted in an objective identification strategy for common clinical isolates belonging to the bacterial speciesStaphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and theStreptococcus millerigroup. Duplicate cultures of 104 different isolates were examined one or more times using1H MRS. A total of 312 cultures were examined. An optimized classifier was developed using a bootstrapping process and a seven-group linear discriminant analysis to provide objective classification of the spectra. Identification of isolates was based on consistent high-probability classification of spectra from duplicate cultures and achieved 92% agreement with conventional methods of identification. Fewer than 1% of isolates were identified incorrectly. Identification of the remaining 7% of isolates was defined as indeterminate.